Compositions and methods for treating and preventing porcine reproductive and respiratory syndrome

ABSTRACT

Disclosed herein are methods and compositions for treating or preventing Porcine reproductive and respiratory syndrome (PRRS) infection in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 13/869,834, filed Apr. 24, 2013, entitled“Compositions and Methods for Treating and Preventing PorcineReproductive and Respiratory Syndrome,” which claims benefit of U.S.Provisional Application No. 61/637,547, filed Apr. 24, 2012, both ofwhich are hereby incorporated herein by reference in their entirety.

BACKGROUND

Porcine reproductive and respiratory syndrome (PRRS) is a chronic viraldisease of pigs worldwide. PRRS is endemic in most pork-producingcountries, and it is responsible for major economic losses to the swineindustry, with an estimated annual loss of $664 million in the US (8).

Since the late 1990s, modified live PRRSV (PRRS-MLV) and killed virusvaccines have been available to control the disease, but neither of themprotects pigs completely against heterologous field viruses (27). Likethe field virus, PRRS-MLV also induces immunosuppression (29, 30).Moreover, there are several reports of reversion of vaccine virus intovirulence leading to severe disease outbreaks (31-34). Although killedPRRSV vaccines are safe, they are poorly immunogenic (35, 36).

Clinical signs of PRRS comprise respiratory and reproductive dysfunctionand the causal agent is PRRS virus (PRRSV) (28). PRRSV establishesdisease by modulating the pig immune system from as early as two daysand continues for several weeks post-infection (14, 15). A particularchallenge presented by PRRSV is the immunosuppression caused by PRRSVattributed to virus mediated reduction in production of importantcytokines (IFN-α, IFN-γ, and TNF-α), associated with increased secretionof interleukin (IL)-10 and transforming growth factor-β (TGF-β), andupregulation of Foxp3⁺ T-regulatory cell (Tregs) population (14). Inaddition, in infected pigs, virus neutralizing (VN) antibodies appeardelayed (3-4 weeks) and also their levels remain low (86). Thus, thereremains a great need in the art to provide for safe and effectiveprotection and treatment of PRRS.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows: (A) Morphology of inactivated PRRSV entrapped PLGAnanoparticles. Scanning electronic photomicrograph of PLGA nanoparticlesprepared by a standard multiple emulsion method. The size of thenanoparticles appears to be variable ranging 200-600 nm. (B) Mucosalvaccination of pigs with inactivated PRRSV entrapped in nanoparticles(Nano-KAg) cleared the viremia by PC 15. Pigs were unvaccinated (n=3) orvaccinated with either killed PRRSV (K-Ag) (n=3) or Nano-KAg (n=3) onceintranasally, and on DPI 21 challenged with PRRSV VR2332 strain. Serumsamples collected on indicated post-challenge day (PC) were analyzed tomeasure PRRSV titers. Each bar represents the average of three pigs±SEM.

FIG. 2 shows enhanced IgA and neutralizing antibody titers in serum andlungs of Nano-KAg vaccinated virus challenged pigs. Analysis wasperformed to determine PRRSV neutralizing antibody response in: (A)serum; (B) lungs by immunofluorescence assay; and (C) anti-PRRSV IgAantibody response in the lung lysate by ELISA. Each bar in the graphrepresents average VN titer or optical density value from threepigs±SEM. Alphabet ‘a’, ‘b’ and ‘c’ represents the statisticalsignificant difference (p<0.05) between unvaccinated vs K-Ag,unvaccinated vs Nano-KAg, and K-Ag vs Nano-KAg pigs, respectively.

FIG. 3 shows cytokine response in serum and lungs of pigs intranasallyvaccinated with Nano-KAg vaccine. Serum samples collected at indicatedPCs were analyzed for cytokines, (A) IFN-γ and (B) TGF-β by ELISA. Lunglysates prepared on the day of necropsy (PC 15) were analyzed for: (C)IFN-γ; (D) IL-6; (E) IL-10; (F) TGF-β; and (G) IL-4 by ELISA. Each baror data point in the graph represents average VN titer or opticaldensity values from three pigs±SEM. Alphabet ‘a’, ‘b’ and ‘c’ representsthe statistical significant difference (p<0.05) between unvaccinated vsK-Ag, unvaccinated vs Nano-KAg, and K-Ag Vs Nano-KAg pigs, respectively.

FIG. 4 shows analysis of PRRSV specific recall cytokine response.Indicated mononuclear cells were restimulated and the harvested culturesupernatant was analyzed for cytokines by ELISA: (A to D) IFN-γ; (E toH) IL-12; (I and J) IL-6; and (K and L) TGF-β. Cytokines secreted byimmune cells cultured in the absence of inactivated PRRSV was subtractedfrom the test values. Each bar in the graph represents average cytokineamount from three pigs±SEM. Alphabet ‘a’, ‘b’ and ‘c’ represents thestatistical significant difference (p<0.05) between unvaccinated vsK-Ag, unvaccinated vs Nano-KAg, and K-Ag vs Nano-KAg pigs, respectively.

FIG. 5 shows cytometric analysis of CD and CD8 positive T cell subsets.Indicated mononuclear cells were immunostained to analyze the frequencyof immune cells: (A to D) CD3⁺ cells; (E to H) CD8⁺ T cells; (I to L)CD4⁺ T cells; and (M to P) CD4^(+CD)8⁺ T cells. Each bar in the graphrepresents average percent of immune cells from three pigs±SEM. Alphabet‘a’, ‘b’ and ‘c’ represents the statistical significant difference(p<0.05) between unvaccinated vs K-Ag, unvaccinated vs Nano-KAg, andK-Ag Vs Nano-KAg pigs, respectively.

FIG. 6 shows cytometry analysis of innate and regulatory T cells.Indicated mononuclear cells were immunostained to determine thefrequency of immune cells: (A to D) γδ T cells; (E to H) myeloid cells;(I to K) Dendritic cells rich fraction; and (L, M and N) Tregs. Each barin the graph represents average percent of immune cells from threepigs±SEM. Alphabet ‘a’, ‘b’ and ‘c’ represents the statisticalsignificant difference (p<0.05) between unvaccinated vs K-Ag,unvaccinated vs Nano-KAg, and K-Ag vs Nano-KAg pigs, respectively.

FIG. 7 shows characterization of Nano-KAg in pig alveolar Mφs in vitro.(A) Scanning electronic photomicrograph of PRRSV Ags entrapped PLGAnanoparticles prepared by a standard multiple emulsion method. Confocalmicroscopy pictures: (B) Mφs were treated with killed PRRSV (K-Ag),Nano-KAg, or infected with PRRSV and then treated with PRRSV N′ proteinspecific mAb followed by Alexa 488 anti-mouse antibody; Co-localizationof PRRSV N protein in endosomes of Mφs, (C) Nano-KAg treated and (D)PRRSV infected. (E) Upregulation of CD80/86 in myeloid cells (CD172⁺)treated with Nano-KAg. Each bar represents average percent of Mφspositive for CD80/86 marker in mock, K—Ag and Nano-KAg treated BAL cellsharvested from three pigs+/−SEM. Asterisk represents the statisticalsignificant difference (p<0.05) between Nano-KAg and K-Ag received piggroups. (F) Representative histogram showing the presence of PRRSV-Nprotein in Mφs treated as indicated. Similar results were obtained inthree independent trials.

FIG. 8 shows Nano-KAg elicited enhanced innate and suppressed regulatoryresponse in a pre-challenge study. Pigs were unvaccinated or vaccinatedwith either K-Ag or Nano-KAg once intranasally. Immunostained to analyzethe frequency of immune cells: (A) NK cells, (B) Dendritic cells, (C) γδT cells, (D) Th/memory cells, (E) CD8⁺ T cells in Lung MNC; and (H) γδ Tcells and (I) Dendritic cells in PBMC. Harvested culture supernatantsfrom restimulated immune cells were analyzed for cytokines (F) IL-10 and(G) IL-6 in lung MNC; and (J) IL-10 in PBMC; also (K) IFN-α in serum byELISA. Each bar represents the average amounts from three pigs±SEM.Asterisk represents the statistical significant difference (p<0.05)between Nano-KAg and K-Ag received pig groups.

FIG. 9 shows reduced lung pathology and viral load in Nano-KAgvaccinated MN184 challenged pigs. Pigs were unvaccinated or vaccinatedwith either K-Ag or Nano-KAg once intranasally, challenged with PRRSVMN184 strain at PID 21 and euthanized at DPC 15. (A) A representativepig lung H&E picture from indicted pig group. (B) A representative lungimmunohistochemistry (IHC) picture from indicted pig group showing PRRSVN antigen positive cells (asterisk in the magnified image) stained byNova red. (C) Gross lung lesions were graded based on percentage of thelung area affected and severity of inflammatory pathology. (D) PRRSV Nantigen positive cells in IHC were counted in 10 random fields from eachpig. The PRRSV titer in fluorescence foci units at indicated DPC in (E)serum, and (F) in the lungs at DPC 15 was determined byimmunofluorescence assay. Each bar represents average values from threepigs±SEM. Asterisk represents the statistical significant difference(p<0.05) between Nano-KAg and K-Ag received pig groups. A similar trendin results was obtained in an independent second trial.

FIG. 10 shows enhanced PRRSV specific IgA and neutralizing antibodyresponse in Nano-KAg vaccinated MN184 strain challenged pigs. Pigs wereunvaccinated or vaccinated with either K-Ag or Nano-KAg onceintranasally, challenged with PRRSV MN184 strain at PID 21 andeuthanized at DPC 15. Anti-PRRSV IgA antibody response in (A) lungs, (D)serum, and (G) nasal swabs; and IgG antibody response in (B) lungs, (E)serum, and (H) nasal swabs was determined by ELISA. PRRSV neutralizingantibody response in (C) lungs and (F) serum was determined byimmunofluorescence assay. Each bar represents average optical densityvalue or VN titer from three pigs±SEM. Asterisk represents thestatistical significant difference (p<0.05) between Nano-KAg and K-Agreceived pig groups. A similar trend in results was obtained in anindependent second trial.

FIG. 11 shows Nano-KAg elicited enhanced innate immune response in thelungs of pigs. Pigs were unvaccinated or vaccinated with either K-Ag orNano-KAg once intranasally, challenged with PRRSV MN184 strain at PID 21and euthanized at DPC 15. Lung homogenate was analyzed for the cytokine(A) IFN-α by ELISA. Lung MNC were analyzed for (B) NK cells, (D) γδ Tcells, (E) CD4⁺ T cells, and (F) CD8⁺ T cells by flow cytometry. (C) NKcells present in the lung MNC were analyzed from cytotoxic function byLDH assay. Each bar or data point in the graph represents average valuesfrom three pigs±SEM. Asterisk represents the statistical significantdifference (p<0.05) between Nano-KAg and K-Ag received pig groups. Asimilar trend in results was obtained in an independent second trial.

FIG. 12 shows a reduction in the immunosuppressive response in Nano-KAgvaccinated MN184 challenged pig lungs. Pigs were unvaccinated orvaccinated with either K-Ag or Nano-KAg once intranasally, challengedwith PRRSV MN184 strain at PID 21 and euthanized at DPC 15. Lung MNCwere analyzed for (A) Tregs population by flow cytometry. Lunghomogenates were analyzed for (B) IL-10, (C) TGF-β, and (D)(IFN-α.Harvested culture supernatants from restimulated lung MNC were analyzedfor cytokines (E) IL-10, (F) TGF-β, and (G) IFN-γ by ELISA. Each barrepresents average values from three pigs±SEM. Asterisk represents thestatistical significant difference (p<0.05) between Nano-KAg and K-Agreceived pig groups. A similar trend in results was obtained in anindependent second trial.

FIG. 13 shows an increased PRRSV specific recall cytokine response inPBMC and TBLN of Nano-KAg vaccinated MN184 strain challenged pigs. Pigswere unvaccinated or vaccinated with either K-Ag or Nano-KAg onceintranasally, challenged with PRRSV MN184 strain at PID 21 andeuthanized at DPC 15. Harvested culture supernatants from restimulatedPBMC and TBLV MNC were analyzed for cytokines by ELISA: (A & E) IL-10,(B) TGF-β, (C & F) IFN-γ, and (D & G) IL-6. Each bar represents averagevalues from three pigs±SEM. Asterisk represents the statisticalsignificant difference (p<0.05) between Nano-KAg and K-Ag received piggroups. A similar trend in results was obtained in an independent secondtrial.

FIG. 14 shows an estimation of PRRSV specific IgA antibody production inbronchoalveolar lavage fluid of pigs against indicated PRRSV structuralproteins and total viral protein by ELISA.

FIG. 15 shows an estimation of PRRSV specific total IgG antibody titerin the blood against total PRRSV protein by ELISA. PC—post-challengeday; Vacci.—1st vaccination; Boost—Booster dose.

FIG. 16 shows an estimation of PRRSV specific neutralization titers(VNT) in the lungs of pigs by immunofluorescence assay.

FIG. 17 shows an estimation of PRRSV specific neutralization titers(VNT) in the blood samples of pigs by immunofluorescence assay.PC—post-challenge day; Vacci.—1st vaccination; Boost—Booster dose.

FIG. 18 shows an estimation of the frequency of IFN-γ secreting cells inthe lungs of pigs specific to indicated PRRSV structural proteins andtotal viral protein determined by ELISPOT assay.

FIGS. 19 (A and B) shows the determination of PRRSV RNA copy number inthe lungs by quantitative real time-PCR (qRT-PCR) and (C & D)determination of infective PRRSV titer in the lungs of pigs determinedby immunofluorescence assay.

FIG. 20 shows a determination of infective PRRSV load in the bloodsamples of pigs determined by immunofluorescence assay.

FIG. 21 shows a determination of infective PRRSV titer in the lungs ofpigs by immuno-fluorescence assay. A & B are the negative and positivecontrol, and C to H are representative pictures of viral load in lunghomogenate of indicated pigs group (n=3) of 500 μg/pig vaccine dosecategory.

FIG. 22 shows a summary of immune responses and virus clearance inNanoparticle entrapped PRRSV K-Ag+Mtb WCL vaccinated and heterologousPRRSV challenged pigs.

FIG. 23 shows a schematic of anti-PRRSV immunity in pigs vaccinatedintranasally with PLGA nanoparticle-entrapped PRRSV vaccine andchallenged with a heterologous virus.

FIGS. 24A, 24B and 24C show the characterization of PRRSV KAg entrappedPLGA nanoparticles (NP-KAg). (A) The pictures were taken by PhilipsXL30-FEG SEM at 20 kV with 30,000× magnification. (B) PRRSV Ags releaseprofile from NP-KAg was performed under normal physiological conditions.(C) Uptake of NP-KAg by pig PAM cells at indicated time pointpost-treatment. Each immunofluorescence picture is a representative ofeach treatment condition: (i) KAg; (ii) NP-KAg; (iii) Sham NPs; (iv)cells control; (v) PRRSV (VR2332) infected. Similar results wereobtained in other two independent experiments.

FIGS. 25A-25F show the significant increase in PRRSV specific antibodiesin pigs vaccinated with adjuvanted NP-KAg. Pigs were vaccinated orunvaccinated with indicated vaccine and adjuvant combination andchallenged with PRRSV MN184. The lung and blood samples collected atindicated PCs were analyzed for virus specific IgA (A & C) and IgG (B,D, E & F) by ELISA: (A&B) BAL fluid; (C, D) lung homogenate; (E, F)plasma samples. Each bar and each symbol in the line graph indicate theaverage value of three pigs±SEM. Asterisk and lowercase alphabetsindicate statistically significant difference between indicated groupsas described in methods. A similar trend in result was obtained inanother independent experiment.

FIGS. 26A-26F show the enhanced heterologous and heterogenotype PRRSVneutralizing antibody (VN) titers in pigs vaccinated with adjuvantedNP-KAg. Pigs were vaccinated and challenged as described in figurelegend 25. The lung homogenate (A, B, E-H) and plasma (C, D) samplescollected at indicated PCs were analyzed for PRRSV VN titers: (A-D) VNtiters against challenged PRRSV (MN184); (E, F) PRRSV (1-4-4); (G, H)PRRSV (SD03-15). Each bar and each symbol in the line graph indicate theaverage value of three pigs±SEM. Asterisk and lowercase alphabetsindicate statistically significant difference between indicated groupsas described in methods.

FIGS. 27A-27I show the enhanced production of Th1-Th2 and suppressedimmunosuppressive cytokines in pigs vaccinated with adjuvanted NP-KAgvaccine (500 μg/pig category). Pigs were vaccinated and challenged asdescribed in figure legend 25. (A) Lung MNCs were restimulated withkilled MN184 Ags and the frequency of IFN-γ secreting cells (ISCs) wasmeasured by ELISPOT. Lung homogenates were analyzed for: (B) IFN-γ; (D)IL-12; (C) IL-6; (E) TGF-β; (F) IL-10 by ELISA. PBMC were restimulatedwith killed MN184 Ags and the supernatant was analyzed for: (G) IL-4;(H) IL-6; (I) IL-10 by ELISA. Each bar indicates the average number ofISCs per million LMNC or average of cytokines from three pigs±SEM.Asterisk indicates statistically significant difference between theindicated pig groups. A similar trend in result was obtained in anindependent second experiment.

FIGS. 28A-28R shows the significantly increased IFN-γ secretinglymphocyte subsets and APCs in pigs vaccinated with adjuvanted NP-KAgvaccine (500 μg/pig dose). Pigs were vaccinated and challenged asdescribed in figure legend 25. LMNC (A-I) and PBMC (J-R) wererestimulated with MN184 Ags and immunostained using indicated cellsurface markers and intracellular IFN-γ and analyzed by Flow cytometry.Frequency of total IFN-γ⁺ cells (Ai, Ji) and a representative histogramof IFN-γ⁺ cells (Aii and Jii) present in the LMNC and PBMC,respectively. The dotted line: isotype control and solid line: IFN-γ⁺specific staining IFN-γ⁺ lymphocyte subsets:CD4⁺IFN-γ⁺ (B, K);CD8⁺IFN-γ⁺ (C, L); CD4⁺CD8⁺IFN-γ⁺ (D, M); γδ⁺IFN-γ⁺ (E, N); NK (CD56⁺)(F, O); and CD56+IFN-γ⁺ cells (G, P) present in LMNC and PBMC wereanalyzed at PC 15. Also APCs population: MΦs rich population(CD172⁺CD163⁺SLA-II⁺) (H, G); and DCs rich population(CD172⁺CD11c⁺SLA-II⁺) (I, R) were analyzed. Each bar indicates theaverage frequency of indicated cells from three pigs±SEM. Asteriskindicates statistically significant difference between indicated piggroups.

FIGS. 29A-29K shows the complete clearance of replicating PRRSV from thelungs and blood of pigs vaccinated with adjuvanted NP-KAg (500 μg/pigdose) vaccine. Pigs were vaccinated and challenged as described infigure legend 2. (A, B) BAL fluid, (E, F) lung homogenate, and (I, J)plasma samples collected at indicated PCs were analyzed for the presenceof replicating PRRSV titer by indirect immunofluorescence assay. PRRSVviral RNA copy numbers in BAL fluid (C, D) and lung homogenate (G, H)were analyzed by qRT-PCR. (K) Representative images of H&E stained lungsections. Each bar or symbol indicates the mean viral titer or viral RNAcopy number of three pigs±SEM. Asterisk or lowercase alphabet indicatesstatistically significant difference between indicated pig groups asdescribed in methods.

FIGS. 30A-30F show the significantly increased PRRSV structural (GP5, M,and N) proteins specific IgG in the blood of pigs vaccinated withadjuvanted NP-KAg. Pigs were vaccinated or unvaccinated with indicatedvaccine and adjuvant combination and challenged with PRRSV MN184. Bloodsamples collected at indicated days were analyzed for IgG titers againstGP5 (A, B), M (C, D) and N (E, F) proteins by ELISA. Each symbolindicates the mean IgG titer±SEM of three pigs of the indicated group.Lowercase alphabet indicate statistically significant (p<0.05)difference between two indicated pig groups as described in materialsand methods. A similar trend in result was obtained in an independentsecond experiment.

FIGS. 31A-31F shows the high avidity PRRSV specific antibodies producedin pigs vaccinated with adjuvanted NP-KAg vaccine. Pigs were vaccinatedor unvaccinated with indicated vaccine and adjuvant combination andchallenged with PRRSV MN184. The lung and blood samples were analyzedfor avidity of PRRSV specific IgA: (A, B) BAL fluid; (C, D) lunghomogenate; (E, F) PRRSV specific IgG in plasma samples by avidityELISA. Each symbol indicates the mean percent retained absorbancecompared to control (NH₄CN at 0 M=100% absorbance)±SEM of three pigs.OD405 at 0 M indicate the mean OD±SEM of three pigs of the indicatedgroup at 0M or no treatment of Ammonium thiocyanate. Lowercase alphabetindicates statistically significant (p<0.05) difference between the twoindicated pig groups as described in materials and methods. A similartrend in result was obtained in an independent second experiment.

FIGS. 32A and 32B show increased levels of PRRSV specific IgG2 (Th1) andIgG1 (Th2) antibodies and Th1-Th2 balanced response in pigs vaccinatedwith adjuvanted NP-KAg vaccine. Pigs were vaccinated or unvaccinatedwith indicated vaccine and adjuvant combination and challenged withPRRSV MN184. The lung homogenate and plasma samples were analyzed forPRRSV specific IgG1 and IgG2 isotype specific antibodies by ELISA. (A)Each bar indicates the average OD value of three pigs±SEM. (B) Ratio ofIgG1:IgG2 denotes Th1 or Th2 biased response. Each bar indicates theaverage ratio and the trend line indicates the cut-off ratio. Ratioof >1 and <1 indicates Th2 and Th1 biased response, respectively.Lowercase alphabet indicates statistically significant (p<0.05)difference between two indicated groups of pigs as described inmaterials and methods. A similar trend in result was obtained in anindependent second experiment.

FIGS. 33A-33I show the enhanced production of Th1-Th2 and suppressedimmunosuppressive cytokines in pigs vaccinated with adjuvanted NP-KAgvaccine (100 μg/pig category). Pigs were vaccinated and challenged asdescribed in figure legend 24. (A) Lung MNCs were restimulated withkilled MN184 Ags and the frequency of IFN-γ secreting cells (ISCs) wasmeasured by ELISPOT. Lung homogenates were analyzed for: (B) IFN-γ; (D)IL-12; (C) IL-6; (E) TGF-β; (F) IL-10 by ELISA. PBMC were restimulatedwith killed MN184 Ags and the supernatant was analyzed for: (G) IL-4;(H) IL-6; (I) IL-10 by ELISA. Each bar indicates the average number ofISCs per million LMNC or average of cytokines from three pigs±SEM.Asterisk indicates statistically significant difference between theindicated pig groups. A similar trend in result was obtained in anindependent second experiment.

FIGS. 34A-34R shows the significantly increased IFN-γ secretinglymphocyte subsets and APCs in pigs vaccinated with adjuvanted NP-KAgvaccine (100 μg/pig dose). Pigs were vaccinated and challenged asdescribed in figure legend 25. LMNC (A-I) and PBMC (J-R) wererestimulated with MN184 Ags and immunostained using indicated cellsurface markers and intracellular IFN-γ and analyzed by Flow cytometry.Frequency of total IFN-γ⁺ cells (Ai, Ji) and a representative histogramof IFN-γ+ cells (Aii and Jii) present in the LMNC and PBMC,respectively. The dotted line: isotype control and solid line: IFN-γ⁺specific staining. IFN-γ+ lymphocyte subsets:CD4+IFN-γ⁺ (B, K);CD8+IFN-γ⁺ (C, L); CD4⁺CD8⁺IFN-γ⁺ (D, M); γδ⁺IFN-γ⁺ (E, N); NK (CD56⁺)(F, O); and CD56⁺IFN-γ⁺ cells (G, P) present in LMNC and PBMC wereanalyzed at PC 15. Also APCs population: MΦs rich population(CD172⁺CD163⁺SLA-II⁺) (H, G); and DCs rich population(CD172⁺CD11c⁺SLA-II⁺) (I, R) were analyzed. Each bar indicates theaverage frequency of indicated cells from three pigs±SEM. Asteriskindicates statistically significant difference between indicated piggroups.

FIGS. 35A and 35B show that NP-KAg (or PLGA-NanoPRRS) significantlyreduced the viral load in the lungs and blood. Pigs were vaccinated asindicated (100 or 500 μg/pig dose) and challenged with a virulenthetrologus PRRSV MN184. PRRSV titers were determined by the indirectimmunofluorescence assay: (A) lung lysate samples (titer in each gram oflung tissue); (B) blood samples (in each mL of plasma). Each bar orsymbol indicates the average titer of three pigs±SEM.Asterisks/alphabets indicate statistically significant (p<0.5)difference in results between group 6 pigs with other tested groups.

FIGS. 36A and 36B show that NP-KAg (or PLGA-NanoPRRS) significantlyincreased the IFN-γ response it he pig lungs. Pigs were vaccinated asindicated and challenged with a virulent heterologous PRRSV MN184. LungMNCs collected at PC 15 were restimulated with killed PRRSV Ags toanalyze: (A) IFN-γ⁺ spots by ELISPOT assay; (B) IFN-γ⁺ lymphocytes byflow cytometry. Each bar indicates the average percent value of threepigs±SEM. Asterisks indicate statistically significant (p<0.05)different in results between group 6 pigs with other tested groups.

FIGS. 37A and 37B show that NP-KAg (or PLGA-NanoPRRS) significantlyincreased the PRRSV neutralization (VN) titers. Pigs were vaccinated asindicated (100 or 500 μg/pig dose) and challenged with a virulenthetrologus PRRSV MN184. Lung lysates prepared at PC 15 and plasmasamples collected at indicated days were analyzed for VN titers againstPRRSV MN184 in: (A) lung lysate and (B) plasma samples. Each bar orsymbol indicates statistically significant (p<0.05) difference inresults between group 6 pigs and other tested groups.

FIGS. 38A and 38B show that NP-KAg (or PLGA-NanoPRRS) significantlyincreased the avidity of PRRSV specific antibodies. Pigs were vaccinated(500 μg dose) as indicated and challenged with a virulent heterologousPRRSV MN184. Plasma and lung lysate samples collected at PC 15 wereanalyzed for virus specific: (A) IgA and (B) IgG avidity by ELISA. Eachsymbol indicates eh mean percent retained Ag-Ab complex compared tocontrol [Ammonium thiocyanate (NH4CN) at 0 M conc.=100%] from threepigs±SEM. Alphabets indicate statistically significant (p<0.05)difference in results between group 6 pigs and other tested groups.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

All patents, patent applications, and publications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretiesinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is in no way intendedthat an order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. The terminology used in thedescription of the embodiments herein is for describing particularembodiments only and is not intended to be limiting of the embodimentsdisclosed. As used in the description, the singular forms “a,” “an,” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in this disclosureare to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numericalparameters set forth in this disclosure are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims, each numerical parameter should be construed in light of thenumber of significant digits and ordinary rounding approaches.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues described herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe application, data are provided in a number of different formats, andthat these data, represent endpoints, starting points, and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point 15 are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15. It is also understood that each unit betweentwo particular units is also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

The term “subject” means an individual. In one aspect, a subject is amammal such as a primate, and, more preferably, a human. Non-humanprimates include marmosets, monkeys, chimpanzees, gorillas, orangutans,and gibbons, to name a few. The term “subject” also includesdomesticated animals, such as cats, dogs, etc., livestock (for example,cattle (cows), horses, pigs, sheep, goats, etc.), laboratory animals(for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guineapig, etc.) and avian species (for example, chickens, turkeys, ducks,pheasants, pigeons, doves, parrots, cockatoos, geese, etc.). Subjectscan also include, but are not limited to fish (for example, zebrafish,goldfish, tilapia, salmon, and trout), amphibians and reptiles. As usedherein, a “subject” is the same as a “patient,” and the terms can beused interchangeably.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

As used herein, the term “amino acid sequence” refers to a list ofabbreviations, letters, characters or words representing amino acidresidues. The amino acid abbreviations used herein are conventional oneletter codes for the amino acids and are expressed as follows: A,alanine; C, cysteine; D aspartic acid; E, glutamic acid; F,phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L,leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R,arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y,tyrosine.

“Polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules. The terms“polypeptide,” “peptide,” and “protein” can be used interchangeably.

In addition, as used herein, the term “polypeptide” refers to aminoacids joined to each other by peptide bonds or modified peptide bonds,e.g., peptide isosteres, etc. and may contain modified amino acids otherthan the 20 gene-encoded amino acids. The polypeptides can be modifiedby either natural processes, such as post-translational processing, orby chemical modification techniques which are well known in the art.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. The same type of modification can be present in the same orvarying degrees at several sites in a given polypeptide. Also, a givenpolypeptide can have many types of modifications. Modifications include,without limitation, acetylation, acylation, ADP-ribosylation, amidation,covalent cross-linking or cyclization, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphytidylinositol,disulfide bond formation, demethylation, formation of cysteine orpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pergylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,and transfer-RNA mediated addition of amino acids to protein such asarginylation. (See Proteins—Structure and Molecular Properties 2nd Ed.,T. E. Creighton, W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pp. 1-12 (1983)).

As used herein, “isolated polypeptide” or “purified polypeptide” ismeant to mean a polypeptide (or a fragment thereof) that issubstantially free from the materials with which the polypeptide isnormally associated in nature. The polypeptides of the invention, orfragments thereof, can be obtained, for example, by extraction from anatural source (for example, a mammalian cell), by expression of arecombinant nucleic acid encoding the polypeptide (for example, in acell or in a cell-free translation system), or by chemicallysynthesizing the polypeptide. In addition, polypeptide fragments may beobtained by any of these methods, or by cleaving full length proteinsand/or polypeptides.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof.

As used herein, “isolated nucleic acid” or “purified nucleic acid” ismeant to mean DNA that is free of the genes that, in thenaturally-occurring genome of the organism from which the DNA of theinvention is derived, flank the gene. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector, such asan autonomously replicating plasmid or virus; or incorporated into thegenomic DNA of a prokaryote or eukaryote (e.g., a transgene); or whichexists as a separate molecule (for example, a cDNA or a genomic or cDNAfragment produced by PCR, restriction endonuclease digestion, orchemical or in vitro synthesis). It also includes a recombinant DNAwhich is part of a hybrid gene encoding additional polypeptide sequence.The term “isolated nucleic acid” also refers to RNA, e.g., an mRNAmolecule that is encoded by an isolated DNA molecule, or that ischemically synthesized, or that is separated or substantially free fromat least some cellular components, for example, other types of RNAmolecules or polypeptide molecules.

As used herein, “sample” is meant to mean an animal; a tissue or organfrom an animal; a cell (either within a subject, taken directly from asubject, or a cell maintained in culture or from a cultured cell line);a cell lysate (or lysate fraction) or cell extract; or a solutioncontaining one or more molecules derived from a cell or cellularmaterial (e.g. a polypeptide or nucleic acid), which is assayed asdescribed herein. A sample can also be any body fluid or excretion (forexample, but not limited to, blood, urine, stool, saliva, tears, bile)that contains cells or cell components.

As used herein, “prevent” is meant to mean minimize the chance that asubject who has an increased susceptibility for developing PRRSinfection will develop PRRS infection.

The term “plurality” refers to two or more. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Additionally,numerical limitations given with respect to concentrations or levels ofa substance, such as an antigen, are intended to be approximate. Thus,where a concentration is indicated to be at least (for example) 200 pg,it is intended that the concentration be understood to be at leastapproximately (or “about” or “about”) 200 pg.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” Thus, unless the context requires otherwise, the word“comprises,” and variations such as “comprise” and “comprising” will beunderstood to imply the inclusion of a stated compound or composition(e.g., nucleic acid, polypeptide, antigen) or step, or group ofcompounds or steps, but not to the exclusion of any other compounds,composition, steps, or groups thereof.

An “immunogenic composition” is a composition of matter suitable foradministration to a human or animal subject (e.g., in an experimentalsetting) that is capable of eliciting a specific immune response, e.g.,against a pathogen, such as PRRSV. As such, an immunogenic compositionincludes one or more antigens (for example, whole purified virus orantigenic subunits, e.g., polypeptides, thereof) or antigenic epitopes.An immunogenic composition can also include one or more additionalcomponents capable of eliciting or enhancing an immune response, such asan excipient, carrier, and/or adjuvant. In certain instances,immunogenic compositions are administered to elicit an immune responsethat protects the subject against symptoms or conditions induced by apathogen. In some cases, symptoms or disease caused by a pathogen isprevented (or treated, e.g., reduced or ameliorated) by inhibitingreplication of the pathogen following exposure of the subject to thepathogen. In the context of this disclosure, the term immunogeniccomposition will be understood to encompass compositions that areintended for administration to a subject or population of subjects forthe purpose of eliciting a protective or palliative immune responseagainst the virus (that is, vaccine compositions or vaccines).

The term “purification” (e.g., with respect to a pathogen or acomposition containing a pathogen) refers to the process of removingcomponents from a composition, the presence of which is not desired.Purification is a relative term, and does not require that all traces ofthe undesirable component be removed from the composition. In thecontext of vaccine production, purification includes such processes ascentrifugation, dialization, ion-exchange chromatography, andsize-exclusion chromatography, affinity-purification or precipitation.Thus, the term “purified” does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified viruspreparation is one in which the virus is more enriched than it is in itsgenerative environment, for instance within a cell or population ofcells in which it is replicated naturally or in an artificialenvironment. A preparation of substantially pure viruses can be purifiedsuch that the desired virus or viral component represents at least 50%of the total protein content of the preparation. In certain embodiments,a substantially pure virus will represent at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, or at least 95% or more of thetotal protein content of the preparation.

An “isolated” biological component (such as a virus, nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell and/ororganism in which the component occurs or is produced. Viruses and viralcomponents, e.g., proteins, which have been “isolated” include viruses,and proteins, purified by standard purification methods. The term alsoembraces viruses and viral components (such as viral proteins) preparedby recombinant expression in a host cell.

An “antigen” is a compound, composition, or substance that can stimulatethe production of antibodies and/or a T cell response in an animal,including compositions that are injected, absorbed or otherwiseintroduced into an animal. The term “antigen” includes all relatedantigenic epitopes. The term “epitope” or “antigenic determinant” refersto a site on an antigen to which B and/or T cells respond. The “dominantantigenic epitopes” or “dominant epitope” are those epitopes to which afunctionally significant host immune response, e.g., an antibodyresponse or a T-cell response, is made. Thus, with respect to aprotective immune response against a pathogen, the dominant antigenicepitopes are those antigenic moieties that when recognized by the hostimmune system result in protection from disease caused by the pathogen.The term “T-cell epitope” refers to an epitope that when bound to anappropriate MHC molecule is specifically bound by a T cell (via a T cellreceptor). A “B-cell epitope” is an epitope that is specifically boundby an antibody (or B cell receptor molecule). An antigen can also affectthe innate immune response.

An “immune response” is a response of a cell of the immune system, suchas a B cell, T cell, or monocyte, to a stimulus. An immune response canbe a B cell response, which results in the production of specificantibodies, such as antigen specific neutralizing antibodies. An immuneresponse can also be a T cell response, such as a CD4+ response or aCD8+ response. In some cases, the response is specific for a particularantigen (that is, an “antigen-specific response”). An immune responsecan also include the innate response. If the antigen is derived from apathogen, the antigen-specific response is a “pathogen-specificresponse.” A “protective immune response” is an immune response thatinhibits a detrimental function or activity of a pathogen, reducesinfection by a pathogen, or decreases symptoms (including death) thatresult from infection by the pathogen. A protective immune response canbe measured, for example, by the inhibition of viral replication orplaque formation in a plaque reduction assay or ELISA-neutralizationassay, or by measuring resistance to pathogen challenge in vivo.

The immunogenic compositions disclosed herein are suitable forpreventing, ameliorating and/or treating disease caused by infection ofthe virus.

The abbreviation “KAg” stands for killed antigen and represents thekilled or inactivated PRRSV. The inactivated PRRSV comprises one or moreimmunogenic PRRS viral proteins and therefore the inactivated PRRSV canbe considered a killed antigen.

The abbreviation “NP-KAg” stands for nanoparticle-killed antigen. Thisrepresents the nanoparticle encapsulated inactivated PRRSV.

As used herein, the terms “virus-like particle” or “VLP” refer to anonreplicating, viral shell. VLPs are generally composed of one or moreviral proteins associated with viral surface capsid structure, such as,but in case of PRRSV they are not limited to structural proteins GP3,GP4, GP5, and matrix proteins of PRRSV or combinations thereof. VLPs canform spontaneously upon recombinant expression of the protein in anappropriate expression system. VLPs, when administered to an animal, canbe immunogenic and thus can cause a protective or therapeutic immuneresponse in the animal. Methods for producing VLPs are generally knownin the art and discussed more fully below. The presence of VLPsfollowing recombinant expression of viral proteins can be detected usingconventional techniques known in the art, such as by electronmicroscopy, biophysical characterization, and the like. See, e.g., Bakeret al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol.(1994) 68:4503-4505. For example, VLPs can be isolated by densitygradient centrifugation and/or identified by characteristic densitybanding.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Every numerical range given throughoutthis specification will include every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein.

In addition, where features or aspects of the inventions are describedin terms of Markush groups or other grouping of alternatives, thoseskilled in the art will recognize that the invention is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group or other group.

COMPOSITIONS

When a human or non-human animal is challenged by a foreignorganism/pathogen the challenged individual responds by launching animmune response which may be protective. This immune response ischaracterized by the co-ordinated interaction of the innate and acquiredimmune response systems.

The innate immune response forms the first line of defense against aforeign organism/pathogen. An innate immune response can be triggeredwithin minutes of infection in an antigen-independent, butpathogen-dependent, manner. The innate, and indeed the adaptive, immunesystem can be triggered by the recognition of pathogen associatedmolecular patterns unique to microorganisms by pattern recognitionreceptors present on most host cells. Once triggered the innate systemgenerates an inflammatory response that activates the cellular andhumoral adaptive immune response systems.

The adaptive immune response becomes effective over days or weeks andprovides the antigen specific responses needed to control and usuallyeliminate the foreign organism/pathogen. The adaptive response ismediated by T cells (cell mediated immunity) and B cells (antibodymediated or humoral immunity) that have developed specificity for thepathogen. Once activated these cells have a long lasting memory for thesame pathogen.

The ability of an individual to generate immunity to foreignorganisms/pathogens, thereby preventing or at least reducing the chanceof infection by the foreign organism/pathogen, is a powerful tool indisease control and is the principle behind vaccination.

Vaccines function by preparing the immune system to mount a response toa pathogen. Typically, a vaccine comprises an antigen, which is aforeign organism/pathogen or a toxin produced by an organism/pathogen,or a portion thereof, that is introduced into the body of a subject tobe vaccinated in a non-toxic, non-infectious and/or non-pathogenic form.The antigen in the vaccine causes the subject's immune system to be“primed” or “sensitised” to the organism/pathogen from which the antigenis derived. Subsequent exposure of the immune system of the subject tothe organism/pathogen or toxin results in a rapid and robust specificimmune response, that controls or destroys the organism/pathogen ortoxin before it can multiply and infect or damage enough cells in thehost organism to cause disease symptoms.

In many cases it is necessary to enhance the immune response to theantigens present in a vaccine in order to stimulate the immune system toa sufficient extent to make a vaccine effective, that is, to conferimmunity. To this end, additives known as adjuvants (or immunepotentiators) have been devised which enhance the in vivo immuneresponse to an antigen in a vaccine composition.

An adjuvant component can increase the strength and/or duration of animmune response to an antigen relative to that elicited by the antigenalone. A desired functional characteristic of an adjuvant component isits ability to enhance an appropriate immune response to a targetantigen.

Described herein are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Thus,if a class of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited each isindividually and collectively contemplated meaning combinations, A-E,A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed.Likewise, any subset or combination of these is also disclosed. Thus,for example, the sub-group of A-E, B-F, and C-E would be considereddisclosed. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods.

Described herein are compositions comprising an immunogenic porcinereproductive and respiratory syndrome virus (PRRSV) antigen and ananoparticle.

The compositions, immunogenic compositions and vaccines described hereincan further comprise one or more adjuvants. The adjuvant can be anycomposition, pharmacological or immunological agent that modifies theeffect of other agents, such as the antigens described herein. Examplesof adjuvants include, but are not limited to Mycobacterium lysate(including a Mycobacterium tuberculosis whole cell lysate), aMycobacterium smegmatis (including Mycobacterium smegmatis whole celllysate), choleratoxin B subunit, and E. coli heat labile mutant toxin.Other examples of adjuvants include evolutionarily conserved molecules,so called PAMPs, which include liposomes, lipopolysaccharide (LPS),molecular cages for antigen, components of bacterial cell walls, andendocytosed nucleic acids such as double-stranded RNA (dsRNA),single-stranded DNA (ssDNA), and unmethylated CpGdinucleotide-containing DNA. Additional examples of adjuvants, include,but are not limited to are aluminum containing adjuvants that include asuspensions of minerals (or mineral salts, such as aluminum hydroxide,aluminum phosphate, aluminum hydroxyphosphate) onto which antigen isadsorbed. Adjuvants Additional examples of adjuvants, include, but arenot limited to aluminum-(alum-)free adjuvants, which are formulated inthe absence of any such aluminum salts. Alum-free adjuvants include oiland water emulsions, such as water-in-oil, and oil-in-water (andvariants thereof, including double emulsions and reversible emulsions),liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids(such as CpG oligonucleotides), liposomes, Toll-like Receptor agonists(particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists), and variouscombinations of such components.

In some embodiments, the compositions, immunogenic compositions andvaccines described herein can further comprise both Mycobacteriumtuberculosis whole cell lysate, Mycobacterium smegmatis whole celllysate, and Mycobacterium vaccae whole cell lysate.

The compositions, immunogenic compositions and vaccines described hereincan comprise one or more nanoparticles. Examples of nanoparticles (usedinterchangably with the term “nanocarrier”) can be found, for example,in U.S. patent application 20100233251. Examples of nanocarriersinclude, but are not limited to nanocarriers composed of one or morepolymers. In some embodiments, the one or more polymers is a watersoluble, non-adhesive polymer. In some embodiments, polymer ispolyethylene glycol (PEG) or polyethylene oxide (PEO). In someembodiments, the polymer is polyalkylene glycol or polyalkylene oxide.In some embodiments, the one or more polymers is a biodegradablepolymer. In some embodiments, the one or more polymers is abiocompatible polymer that is a conjugate of a water soluble,non-adhesive polymer and a biodegradable polymer. In some embodiments,the biodegradable polymer is polylactic acid (PLA), poly(glycolic acid)(PGA), or poly(lactic acid/glycolic acid) (PLGA). In some embodiments,the nanocarrier is composed of PEG-PLGA polymers.

In some embodiments, the nanocarrier is formed by self-assembly.Self-assembly refers to the process of the formation of a nanocarrierusing components that will orient themselves in a predictable mannerforming nanocarriers predictably and reproducably. In some embodiments,the nanocarriers are formed using amphiphillic biomaterials which orientthemselves with respect to one another to form nanocarriers ofpredictable dimension, constituents, and placement of constituents. Insome embodiments, the nanocarrier is a microparticle, nanoparticle, orpicoparticle. In some embodiments, the microparticle, nanoparticle, orpicoparticle is self-assembled.

In some embodiments, the nanocarrier has a positive zeta potential. Insome embodiments, the nanocarrier has a net positive charge at neutralpH. In some embodiments, the nanocarrier comprises one or more aminemoieties at its surface. In some embodiments, the amine moiety is aprimary, secondary, tertiary, or quaternary amine. In some embodiments,the amine moiety is an aliphatic amine. In some embodiments, thenanocarrier comprises an amine-containing polymer. In some embodiments,the nanocarrier comprises an amine-containing lipid. In someembodiments, the nanocarrier comprises a protein or a peptide that ispositively charged at neutral pH. In some embodiments, the nanocarrieris a latex particle. In some embodiments, the nanocarrier with the oneor more amine moieties on its surface has a net positive charge atneutral pH.

Nanoparticles can aid the delivery of the inactivated PRRSV and/or canalso be immunogenic. Delivery can be to a particular site of interest,e.g. the mucosa. In some embodiments, the nanoparticle can create atimed release of the inactivated PRRSV to enhance and/or extend theimmune response. In some embodiments, the nanoparticle is associatedwith the inactivated PRRSV such that the composition can elicit animmune response. The association can be, for example, wherein thenanoparticle is coupled or conjugated with the inactivated PRRSV. Bycoupled and conjugated is meant that there is a chemical linkage betweenthe nanoparticle and the inactivated PRRSV. In some embodiments, theinactivated PRRSV is entrapped or encapsulated within the nanoparticle.In some embodiments, the inactivated PRRSV is entrapped within thenanoparticle by a water/oil/water emulsion method. In some embodiments,the nanoparticle is poly(lactide co-glycolide) (PLGA). Depending on theratio of lactide to glycolide used for the polymerization, differentforms of PLGA can be obtained and utilized. These forms are typicallyidentified in regard to the monomers' ratio used (e.g. PLGA 75:25identifies a copolymer whose composition is 75% lactic acid and 25%glycolic acid). Different ratios can be used in this invention, e.g.90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, andnumbers above and in between these ratios. Additional examples ofsuitable nanoparticles include chitosin, calcium phosphate, lipids ofvarious bacteria like E. Coli, mycobactera, leptospira and mixturesthereof. In one example, the composition can be derived mixing about 180mg of PLGA to about 5 mg of inactivated PRRSV (or about 36 mg PLGA to 1mg inactivated PRRSV). The entrapment (encapsulation) efficiency ofinactivated PRRSV can vary. In one embodiment the nanoparticle were50-55% entrapped/encapsulated, calculated based on amount of total PRRSVprotein used in the entrapment. Entrapped inactivated PRRSV can beadministered as mixtures of entrapped/encapsulated andunentrapped/unencapsulated antigens or the entrapped/encapsulatedantigens can be further purified.

In some embodiments, the antigen is derived from inactivated or killedPRRSV. In one embodiment, the PRRSV is inactivated or killed by UVlight. Other means of inactivation include chemical, heat, orradioactivity.

Any suitably immunogenic inactivated PRRSV or PRRSV antigen can beutilized in the composition. The complete genome sequence of the PRRSvirus is about 15 kb in size and contains 7 Open Reading Frames (ORF).The six smaller ORF's, 2-7, encode structural proteins associated withthe virion and are examples that can be used in the compositions of thisinvention. Examples of immunogenic antigens include GP2, 3, 4, 5, M 19,N15, mixtures thereof, and the like. The PRRSV antigen can berecombinantly derived.

The disclosed compositions can comprise an inactivated PRRSV that isimmunogenic. The disclosed compositions can also comprise an inactivatedPRRSV that comprises PRRSV antigens. For example, the PRRSV antigen canbe a PRRSV surface glycoprotein.

Disclosed are compositions comprising virus-like particles (VLPs) and ananoparticle. The disclosed compositions can comprise a VLP that isimmunogenic. VLPs resemble viruses, but are non-infectious because theydo not contain any viral genetic material. The expression of viralstructural proteins, such as Capsid, can result in the self-assembly ofVLPs. VLPs can be produced in a variety of cell culture systemsincluding mammalian cell lines, insect cell lines, yeast, and plantcells. For example, the VLP can be produced by a baculovirus or a plantsystem. The VLP can be immunogenic. Any of the disclosed nanoparticlescan be used to entrap the PRRSV VLP. For example, disclosed are PRRSVVLPs entrapped in PLGA nanoparticles.

In some embodiments, the disclosed compositions further contain thelectin Ulex Europaeus Agglutinin-I (UEA). The UEA can be entrapped inthe nanoparticle with the inactivated PRRSV or VLPs and can also besurface-anchored to the nanoparticle. M cells are specializedendothelial cells overlaying the mucosal lymphoid follicles called thefollicle-associated epithlium (FAE) and contain α-1-fucose receptorswhich bind to UEA. Thus, the presence of the UEA can help direct thecomposition or vaccine to mucosal specialized follicle or epithelialcells.

Described herein are vaccines comprising a composition of this inventionin a carrier wherein the vaccine is protective against PRRSV infection.The term “immunogenic carrier” as used herein can refer to a firstpolypeptide or fragment, variant, or derivative thereof which enhancesthe immunogenicity of a second polypeptide or fragment, variant, orderivative thereof. An “immunogenic carrier” can be fused, to orconjugated/coupled to the desired polypeptide or fragment thereof. See,e.g., European Patent No. EP 0385610 B1, which is incorporated herein byreference in its entirety for its teaching of fusing, conjugating orcoupling a polypeptide to a carrier. An example of an “immunogeniccarrier” is PLGA. In some embodiments the vaccine can comprise wholevirus inactivated PRRSV, encapsulated by PLGA, and a carrier. In someembodiments the vaccine can further comprise a Mycobacteriumtuberculosis whole cell lysate.

Methods

Described herein are methods of eliciting an immune response againstPRRSV in a pig comprising administering to the pig a composition of theinvention. The immune response can be protective. The method can furthercomprise administering to the pig virulent PRRSV to monitor the vaccineefficacy. The virulent PRRSV can be the MN184 strain of PRRSV, avirulent and genetically variant strain of PRRSV.

Described herein are methods of reducing reproductive or respiratoryfailure in pigs comprising the steps of: providing a compositionprovided herein; and administering the composition to pigs. The methodcan further comprise administering to the pig virulent PRRSV to monitorthe vaccine efficacy. The virulent PRRSV can for example be the MN184strain of PRRSV. Also described are methods of stimulating an immuneresponse in a pig comprising: administering to said pig a vaccine orcomposition provided herein.

Described herein are methods of administering the compositions andmethods set forth herein for the complete clearance of PRRSV viremia.Also described are methods and compositions that can be used to increase(e.g. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 etc. foldincreased) anti-PRRSV humoral and cell-mediated immune response comparedto killed PRRSV vaccine antigens (K-Ag) in immunized homologous viruschallenged pigs. The methods and compositions described herein can alsobe used to provide a significant increase (e.g. 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30 etc. fold increased) in virus neutralizingantibodies and IgA response in the lungs and blood when compared tokilled PRRSV vaccine antigens (K-Ag) in immunized, homologous viruschallenged pigs. The methods and compositions described herein can alsobe used to provide lung lysate and serum of Nano-KAg vaccinated pigswith higher levels (e.g. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30etc. fold increased) of IFN-γ and IL-12, and lower levels ofimmunosuppressive mediators (IL-10 and TGF-β) (e.g. 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30 etc. fold decreased) compared to control piggroups. The methods and compositions described herein can also be usedto provide mononuclear cells from the lungs, blood, BAL, TBLN, and bloodof Nano-KAg vaccinated pigs having increased frequencies (e.g. 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 etc. fold increased) of CD4⁺,CD8⁺, CD4⁺CD8⁺ T cells, γδ T cells, myeloid cells, and dendritic cellsrich fractions. The methods and compositions can also be used to providea decrease (e.g. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 etc.fold decreased) in Foxp3⁺ T-regulatory cells. The compositions andmethods can also be used to provide intranasal delivery of PLGAnanoparticle-entrapped PRRSV killed vaccine that elicits an immuneresponse at both mucosal and systemic sites sufficient to clear theviremia in pigs.

Also provided are the composition and methods than can be used toprovide protective systemic and mucosal immune responses against PRRSVthat can clear the viremia early post-infection, e.g. three, two, andone week post infection (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30 days).

The vaccine or composition can also be administered at a dose, forexample, of between 100 ug/pig and 1 mg/pig. Other examples includedoses comprising 50 ug/pig and 500 ug/pig. The composition or vaccinecan be administered for example in a single dose, or in two or moredoses. In one embodiment, the two doses are administered at a two weekinterval. The composition or vaccine can, for example, be administeredintranasally. Additional examples of alternative routes of immunizationinclude intramuscular, subcutaneous, intranasal drops, and intranasalaerosol delivery.

The compositions and methods can also be used at a dose of vaccine orimmunogen having less than 1×10⁸ TCID₅₀ of PRRSV. Also provided is adose less than 1×10⁷, 1×10⁶, and 1×10⁵ TCID₅₀ of PRRSV. Furtherdisclosed herein, each dose can be approximately 5×10⁶TCID₅₀ of PRRSV.Also provided are examples of doses between 1×10⁸ TCID₅₀ of PRRSV and1×10⁵ TCID₅₀ of PRRSV, between 1×10⁷ and 1×10⁵TCID₅₀ of PRRSV, between1×10⁶ and 5×10⁶TCID₅₀ of PRRSV. The doses can be derived from UV treatedPRRSV. The doses can be administered as a single reduced viral dose toelicit a protective immune response.

EXAMPLES Example 1 Nanoparticle Encapsulated Killed Porcine Reproductiveand Respiratory Syndrome Virus Vaccine Elicits Adequate Immune Responsein Pigs

In this Example, a PLGA [poly(D,L-lactide-co-glycolide)] nanoparticleentrapped candidate killed PRRSV vaccine (Nano-KAg) was developed andadministered intranasally and evaluated immune correlates in homologousvirus challenged pigs. The results in Nano-KAg vaccinated pigsidentified complete clearance of viremia associated with increasedanti-PRRSV humoral and cell-mediated immune response compared tounvaccinated and killed PRRSV vaccine antigens (K-Ag) immunized,homologous virus challenged pigs. Immunologically, Nano-KAg immunizedpigs had a significant increase in virus neutralizing antibodies and IgAresponse in the lungs and blood. Lung lysate and serum of Nano-KAgvaccinated pigs had higher levels of IFN-γ and IL-12, and lower levelsof immunosuppressive mediators (IL-10 and TGF-β) compared to control piggroups. Mononuclear cells from the lungs, blood, BAL, TBLN, and blood ofNano-KAg vaccinated pigs had increased frequencies of CD4+, CD8+,CD4+CD8+ T cells, γδ T cells, myeloid cells, and dendritic cells richfraction; and conversely a decrease in Foxp3+T-regulatory cells.Overall, the results show that intranasal delivery of PLGAnanoparticle-entrapped PRRSV killed vaccine was safe, and it elicitsadequate immune response at both mucosal and systemic sites sufficientto clear the viremia in pigs.

Material and Methods

Cells, PRRSV and Polymer

Stable Mycoplasma-free MARC-145 cells (37) were used for preparation ofthe vaccine and in vitro assays. Cells were maintained in DMEM (Lonza)with 10% fetal bovine serum (Atlanta Biologicals). The virus infectionmedium was DMEM supplemented with 2% horse serum. For preparation ofnanoparticles, PLGA 50:50 (mol. wt 40-75 kDa), polyvinyl alcohol (mol.wt. 30-70 kDa) (Sigma-Aldrich), Dicholoro methane (Acros Organics), andBCA (bicinchoninic acid) protein assay kit (Pierce) were used.

Animals

Conventional Large White-Duroc crossbred weaned specific-pathogen-freepigs at 3-4 wks of age were housed in a BSL-2 facility at FAHRP, OARDC.The swine herd was confirmed seronegative for PRRSV, porcine respiratorycorona virus, transmissible gastroenteritis virus, and porcine circovirus 2. Piglets blood samples collected on arrival were confirmednegative for PRRSV antibodies. All the animals received food and waterad libitum.

Preparation of Inactivated PRRSV

MARC-145 cell-monolayer was infected with the North American prototypePRRSV strain VR2332 strain (38) at 0.01 MOI (multiplicity of infection),and freeze-thawed thrice when >80% cytopathic effect was seen. Harvestedinfected cell culture fluid was clarified at 2000×g and then subjectedto ultracentrifugation with a 20% sucrose overlay at 100,000×g for 2 hr.Pooled crude viral pellet was suspended in sterile PBS and titrated. Thepellet was UV irradiated (254 nm for 1 hr), confirmed the inactivation,sonicated, protein content was estimated using BCA kit (Pierce), andstored at −70° C. Control antigen was prepared in the same manner usinguninfected MARC-145 cells.

Preparation of PLGA Nanoparticles

Nanoparticles were prepared by a standard double emulsion solventevaporation method (40). Briefly, 15% of PLGA 50/50 (750 mg) wasdissolved in 5 ml of dichloromethane and 100 μl of killed VR2332proteins (5 mg) was added. The mixture was homogenized for 90 secondsusing a Brinkman Polytron homogenizer at 6000 rpm. The homogenizedmixture was added to 60 ml of aqueous solution of polyvinyl alcohol (10%PVA), and homogenized for 5 min. Finally, the preparation was stirredovernight at room temperature (RT) to allow solvent evaporation. Thenanoparticles were washed in distilled water three times and the wetnanoparticles were freeze-dried and stored at 4° C.

Determination of Size, Morphology, and Protein Entrapment Efficiency ofNanoparticles

The size and morphology of nanoparticle was detected using scanningelectron microscopy (Hitachi S-3500N). Briefly, freeze-driednanoparticles were mounted on an adhesive stub coated with gold platinumunder vacuum using an ion coater. The coated specimen was examined underthe microscope at 10 KV. The amount of entrapped inactivated PRRSV inthe nanoparticles was determined as described previously (41).

Pigs and Inoculations

Pigs (n=12) were divided into four groups (n=3 pigs per group). GroupI—mock pigs; Group II—inoculated with normal saline; GroupIII—inoculated with killed VR2332 antigens (K-Ag); Group IV—inoculatedwith nanoparticle-entrapped killed VR2332 antigens (Nano-KAg). Eachvaccine (Nano-KAg and K-Ag) dose was 1 mg of crude viral preparationcontaining equivalent ˜5×10⁶ TCID50 of inactivated virus. Groups II,III, and IV were challenged with PRRSV VR2332 (1×106 TCID50/ml, 2 ml perpig) on day post-immunization (DPI) 21 and euthanized on daypost-challenge (DPC or PC) 15. All the inoculations in this study wereperformed once by intranasal route. Mock-inoculated pigs were euthanizedseparately before virus challenged animals.

Collection of Blood and Lung Samples for Analysis

For evaluation of viremia and for titration of PRRSV specific VNantibodies, 3 to 5 ml of blood samples were collected on DPI 0 and 21,and DPC 7 and 15, and serum samples were aliquoted and kept at −20° C.Lung homogenates were prepared for cytokine and virus evaluation (46,12).

Isolation of PBMC, Lung MNC, BAL, TBLN Cells

Isolation of PBMC, lung-mononuclear cells (lung MNC/LMNC), andtracheobronchial lymph nodes (TBLN) MNC was performed as per thedescribed procedure (20, 21). The airways were lavaged to collect BALcells using sterile ice cold PBS containing EDTA (0.03%) (49).

Virus Titration, Virus-Neutralizing Test (VNT), and Isotype SpecificAntibody Analysis

PRRSV titer and VN antibody titer in serum and in lung lysate wereanalyzed by indirect immunofluorescence assay (IFA) (37, 48). PRRSVspecific IgA antibodies in serum and lung lysate were analyzed by ELISA(95). To eliminate the background activity, non-PRRSV specific antigencoated control plates were also used, blocked and treated with testsamples side-by-side. The OD values obtained from experimental plateswere subtracted from the control plate.

PRRSV Specific Recall/Memory Immune Response

Five million pig PBMC, TBLN MNC, and lung MNC were subjected to ex vivorestimulation in the absence or presence of killed crude PRRSV VR2332antigens (Ags) (50 μg/ml) as described (48), and the harvestedsupernatant was analyzed to measure cytokines.

Analysis of Cytokine Response and Flow Cytometric Analyses of ImmuneCells

Serum samples, harvested culture supernatants, and lung lysates wereanalyzed for Th1 (IFN-γ and IL-12), Th2 (IL-4), pro-inflammatory (IL-6),and immunosuppressive (IL-10 and TGF-β) cytokines by ELISA (48). Amountof cytokines present in the lung lysate was converted to picogram pergram of lung tissue. Flow cytometry analysis was performed to determinethe phenotype and the frequency of different immune cells by amulticolor immunoassay as described (48).

Statistical Analysis

All data were expressed as the mean+/−SEM of three pigs. Statisticalanalyses were performed using one way analysis of variance (ANOVA)followed by post-hoc Tukey's test using GraphPad InStat (softwareversion 5.0 for windows) to establish differences between experimentalgroups. Statistical significance was assessed as P<0.05.

Results

Characterization of Inactivated PRRSV Entrapped Nanoparticles

The size of both sham as well as inactivated PRRSV loaded nanoparticleswas in the range of 200-600 nm (FIG. 1A). The yield of preparednanoparticles was 82.32±3.3% calculated based on amount of PLGA polymerused. The morphology of the nanoparticles containing inactivated PRRSVwas spherical with no surface discontinuity (FIG. 1A). The entrapmentefficiency of inactivated PRRSV in nanoparticles was 50-55%, calculatedbased on amount of total PRRSV protein used in the entrapment.

Viral Load and Humoral Immune Response in Pigs

Nano-KAg vaccinated pigs had reduction in viremia by greater than 1 logat PC 8 and the viremia was completely cleared by DPC 15 compared toboth the control pig groups (FIG. 1B).

Nano-KAg vaccinated pigs had consistently increased levels of VNantibodies in serum with significantly higher levels at PC 0 and 7compared to both the control pig groups (FIG. 2A). Similarly,significantly increased VN antibody titer in the lung lysate of Nano-KAgreceived pigs at PC 15 compared to both the control pig groups wasdetected (FIG. 2B). However, lungs of both K-Ag and Nano-KAg vaccinatedpigs had significantly increased PRRSV specific IgA titer compared tounvaccinated virus challenged animals (FIG. 2C).

Secretion of Increased Th1, Reduced Th2 and Immunosuppressive Cytokinesin Nano-KAg Vaccinated Pigs

The Th1 cytokine, IFN-γ, in serum of unvaccinated pigs was undetectable,and it was at lower levels in serum of K-Ag immunized, virus challengedpigs. However, in Nano-KAg vaccinated pigs, a significantly increasedlevel of IFN-γ at PC 7 and 15 compared to both control groups wasdetected (FIG. 3A). Similarly, Nano-KAg immunized pig lungs hadsignificantly higher levels of IFN-γ secretion than both the control piggroups at PC 15 (FIG. 3C).

K-Ag vaccinated pigs had significantly increased TGF-β levels in serumcompared to other experimental groups at PC 0. Unvaccinated, viruschallenged pigs also had an increased trend in serum TGF-β levels atboth PC 7 and PC-15 (FIG. 3B). However, Nano-KAg vaccinated pigs hadsignificantly reduced TGF-β levels in serum at PC 7 and 15 (FIG. 3B).The proinflammatory cytokine, IL-6, in the lungs was significantlyreduced in Nano-KAg and K-Ag immunized compared to unvaccinated pigs atPC 15. Moreover, reduction in IL-6 in Nano-KAg inoculated pigs wassignificantly lower than K-Ag immunized pigs (FIG. 3D). Both theimmunosuppressive cytokines (IL-10 and TGF-β) and a Th2 cytokine, IL-4,in the lungs of Nano-KAg and K-Ag immunized pigs were significantlyreduced compared to unvaccinated pigs at PC 15 (FIGS. 3 E, F and G).

Enhanced Th1 and Reduced Proinflammatory Recall Cytokines Response inNano-KAg Vaccinated Pigs

In LMNC, PBMC, BAL, and TBLN of Nano-KAg immunized pigs a significantlyincreased secretion of Th1 cytokines (IL-12 and IFN-γ) compared tounvaccinated virus challenged pigs was observed (FIG. 4A to H).Moreover, in PBMC, BAL, and TBLN cultures amount of secreted Th1cytokines in Nano-KAg vaccinated pigs was significantly higher than K-Agreceived pigs (FIG. 4A to H). In K-Ag received pigs the amounts of IFN-γin the culture supernatant of BAL and TBLN cultures, and IL-12 in BALcultures was significantly higher than unvaccinated virus challengedpigs (FIGS. 4CD and G).

Secretion of proinflammatory cytokine, IL-6, in PBMC and LMNC ofNano-KAg immunized pigs was significantly less compared to unvaccinatedand K-Ag immunized pigs (FIGS. 4I and J). The LMNC and BAL-MNC culturesof Nano-KAg immunized pigs secreted significantly less amounts of TGF-βcompared to both the control groups (FIGS. 4 K and L).

Phenotypic Analysis of Different Immune Cells

Frequency of total lymphocyte population (CD3+) in BAL-MNC of Nano-KAgand K-Ag immunized pigs was significantly increased compared tounvaccinated group (FIG. 5C). In the LMNC of Nano-KAg immunized pigs, asignificantly higher frequency of CD4+, CD8+, and CD4+CD8+ T cellscompared to unvaccinated virus challenged pigs was detected (FIGS. 5F,J, and N). In BAL-MNC of Nano-KAg received pigs, a similar significantincrease in lymphocyte subsets was observed. In addition, an increase inCD4+CD8+ T cells was significantly higher than K-Ag vaccinated pigs(FIG. 5K).

In Nano-KAg and K-Ag received pigs, frequency of T cells (TcR1N4⁺CD8⁺)in LMNC, PBMC, BAL, and TBLN-MNC was significantly higher compared tounvaccinated pigs (FIGS. 6ABC and D). In addition, their population inPBMC and BAL-MNC of Nano-KAg pigs was significantly higher than K-Agvaccinated pigs (FIG. 6C). Myeloid cells and DCs rich fraction in PBMCand LMNC of Nano-KAg vaccinated pigs was significantly higher than K-Agvaccinated pigs (FIGS. 6EFI and J). Also in LMNC of both Nano-KAg andK-Ag vaccinated pigs, myeloid cells and DCs rich fraction wassignificantly higher than unvaccinated pigs (FIGS. 6E and I). However,no alterations in myeloid cells and DCs rich fraction in BAL and TBLN(FIGS. 6GH and K) were found.

Frequency of Tregs in LMNC was significantly lesser in Nano-KAgimmunized compared to unvaccinated pigs (FIG. 6L). While in pigsimmunized with K-Ag, a significantly higher frequency of Tregs in LMNCcompared to unvaccinated group was detected (FIG. 6L). Tregs frequencyin PBMC in Nano-KAg immunized pigs compared to either control groups wassignificantly reduced (FIG. 6M). Also in TBLN of Nano-KAg immunized pigssignificantly reduced population of Tregs compared to K-Ag immunizedpigs was detected (FIG. 6N).

In spite of regular use of existing PRRSV vaccines prevention andcontrol of the disease has remained as a challenge. There are outbreaksof PRRS in live vaccine immunized pig herd associated with isolation ofreverted virus in unvaccinated sows in the same farm premises wasreported (32, 96, 97).

PRRSV neutralizing antibodies protect pigs against viremia, virusreplication in the lungs, transplacental spreading of the virus, andreproductive failure (98, 99). But the available killed PRRSV vaccinesinduce negligible VN antibody titers (35, 36, 100). A study hasdemonstrated that it is possible to elicit increased VN antibodies to akilled PRRSV vaccine by inactivating the virus using UV or binaryethylenimine (BEI), and co-administering the vaccine with a potentadjuvant (100). Each dose of UV or BEI treated vaccine had 1×10⁸ TCID50of the sucrose gradient purified PRRSV (100). As disclosed herein, eachvaccine was having approximately 5×10⁶ TCID50 of UV-treated PRRSV. Thisshows the advantage of using nanoparticle-mediated mucosal delivery ofkilled PRRSV vaccine in pigs, wherein a single reduced viral doseelicited protective immune response.

Incomplete anti-PRRSV immunity in immunized and infected pigs wasassociated with low levels of IFN-γ production (77). Enhanced IFN-γresponse and antigen specific T-cell response (CD4+ and CD8+ T cells) isthe hallmark of anti-PRRSV immunity (104, 105). A significant increasein IFN-γ levels and CD4+ and CD8+ T cell population in the lungs andblood of Nano-KAg vaccinated pigs were detected. T lymphocyte subsetwith CD4+CD8+ phenotype were abundant in pigs and they possess memory,T-helper, and cytolytic properties (106). In Nano-KAg immunized pigs,there was a significant increase in the population of CD4⁺CD8⁺ T cellsin the lungs and blood. In pigs infected with an European strain ofPRRSV a negative correlation between the frequency of CD4⁺CD8⁺ T cellsand viremia was reported at 14 and 24 days post-infection, indicatingtheir protective role in anti-PRRSV immunity (107). Recall cytokineresponses in LMNC, PBMC, BAL, and TBLN-MNC of Nano-KAg immunized pigsrevealed secretion of significantly increased Th1 cytokines (IFN-γ andIL-12). The cytokine, IL-12, plays an important role in host defenseagainst viral infections (108).

A significant reduction in immunosuppressive cytokines (IL-10 and TGF-β)in the lungs of Nano-KAg immunized pigs was associated with enhancedIFN-α production both in the lungs and blood. In pigs, IL-10 reportedlyinhibits IFN-γ production by T cells (109), and its immunosuppressivefunction with reduced IFN-γ production was reported in PRRSV infectedpigs (77, 78). Infiltration of Tregs in infected pig lungmicroenvironment contributes to secretion of high levels of IL-10 andTGF-β (75). An increased frequency of Tregs in PRRSV infected pigs wasreported (48, 110); but in Nano-KAg (but not K-Ag) immunized pigs areduction in Tregs population in LMNC, PBMC, and TBLN was observed. Pigspossess a relatively large population of γδ T cells, and they areconsidered as an important innate immune cell at mucosal sites (111). InPRRSV infected pigs γδ T cells secrete IFN-γ (73). In Nano-KAgvaccinated pigs, there was an increased frequency of γδ T cells in thelungs, blood, and TBLN, indicating the protective role played by γδ Tcells in PRRSV immunity.

In conclusion, the nanoparticle-mediated intranasal delivery of killedPRRSV vaccine elicits protective systemic and mucosal immune responsesagainst PRRSV, and can clear the viremia early post-infection.

Example 2 Biodegradable Nanoparticle-Entrapped Vaccine InducesCross-Protective Immune Response Against a Virulent HeterologousRespiratory Viral Infection in Pigs

Material and Methods

Cells, PRRSV and Biodegradable Polymer

A stable Mycoplasma-free MARC 145 cells (African Green monkey kidneycell line) which supports the growth of PRRSV (37) were used to preparePRRSV stocks, killed viral Ags, and for immunological assays. Cells weremaintained in Dulbecco's minimum essential medium (DMEM, Lonza) with 10%fetal bovine serum (Atlanta Biologicals) at 37° C. with 5% CO2. Forvirus infection and titration, the infection medium used was DMEMsupplemented with 2% horse serum. The North American prototype PRRSVstrain VR2332 (was used to prepare the vaccine, and for viral challengestudies an antigenically highly divergent virulent heterologous PRRSVstrain MN184 (38) was used. For preparation of nanoparticles, PLGA [poly(DL-lactide-coglycolide) 50:50, mol. wt. 40-75 kDa), polyvinyl alcohol(mol. wt. 30-70 kDa) (Sigma-Aldrich), Dicholoro methane (AcrosOrganics), and BCA protein assay kit (Pierce) were used.

Preparation of PRRSV Ags

MARC-145 cell-monolayer (>90% confluent) was infected with the VR2332strain (38) at 0.001 MOI (multiplicity of infection) in roller bottlesor T150 tissue culture flasks, and freeze-thawed three times after cellsshowed more than 80% cytopathic effect (approximately 3-4 days).Harvested infected cell culture fluid was clarified at 2000×g for 15minutes and ultracentrifuged with a 20% sucrose overlay at 100,000×g for2 hr at 40° C. Pooled crude viral pellet was suspended in sterile PBSand titrated to determine the viral titer, and UV irradiated (254 nm for1 hr) to inactivate the virus, sonicated (probe sonicator at 80%amplitude, 30 seconds for 3 cycles), and the protein content wasestimated using BCA kit (Biorad). Crude virus pellet was aliquoted andstored at −70° C. Control antigen was prepared similarly usinguninfected MARC-145 cells.

Preparation of PLGA Nanoparticle-Entrapped Killed PRRSV Vaccine(Nano-KAg)

Nanoparticles were prepared using standard double emulsion solventevaporation technique (40, 39). Briefly, 15% of PLGA 50/50 (750 mg) wasdissolved in 5 ml of dichloromethane, and 100 μl of killed VR2332proteins (5 mg) was added. The mixture was homogenized using a BrinkmanPolytron homogenizer at 6000 rpm for 90 seconds. The homogenized mixturewas added to aqueous solution of polyvinyl alcohol (10% PVA), andhomogenized for 5 min. Finally, the preparation was stirred at roomtemperature (RT) overnight to allow solvent evaporation; and thenanoparticles were washed in distilled water three times bycentrifugation at 11,000×g, freeze-dried and stored at 4° C.

Determination of Entrapment Efficiency of PRRSV Proteins inNanoparticles

The amount of entrapped PRRSV protein in the nanoparticles wasdetermined as described previously (41). Briefly, freeze-dried Nano-KAg(10 mg) was treated with 0.1N NaOH for 1 hr at 37° C., vortexed and theharvested supernatant was collected and analyzed for proteinconcentration, including a series of BSA standards prepared in 0.1 NNaOH in a BCA protein assay kit (Pierce, USA).

Determination of Morphology of PLGA Nanoparticles

The size and shape of nanoparticles was determined by scanning electronmicroscopy (Hitachi S-3500N). Briefly, freeze-dried nanoparticles weremounted on an adhesive stub coated with gold-platinum under vacuum usingan ion coater, and examined under the microscope at 10 KV.

Characterization of Nano-KAg by Confocal Microscopy

Bronchoalveolar lavage fluid (BAL) collected from three 4-6 weeks oldhealthy SPF pigs was processed to isolate mononuclear cells (BAL-MNC)(42). BAL-MNC plated at a concentration of 1×10⁶ per ml in a 24 wellplate containing poly-L-lysine coated cover slips were incubated at 37°C. in 5% CO2 incubator for 1 hr. Non-adherent cells were aspirated andthe coverslips with adherent cells were washed gently with PBS and usedin the study. Freeze-dried Nano-KAg containing different concentrationsof PRRSV proteins (0.2 μg/ml) was suspended in DMEM containing 10% FBSand added into a plate containing BAL cells and incubated for 3 hr at37° C. BAL-MNC, either uninfected or infected with PRRSV (MN184 strain)at 0.1 MOI for 12 hr was included in the assay. Cells were fixed in 3%paraformaldehyde for 15 min on ice, permeabilized (0.1% Triton X-100 for15 min) and blocked (PBS containing 5% BSA and 0.2% triton X-100 for 1hr at RT). Subsequently, cells were treated with anti-PRRSV nucleocapsidspecific mAb SDOW17 (Rural Technologies, Inc.) and early endosomecross-reactive anti-pig human antibody (Santa-Cruz) in PBS containing 1%BSA and 0.1% triton X-100 for 1 hr at RT. Followed by treatment withgoat anti-mouse IgG Alexa Flour488 and donkey anti-goat Alexa flour 633(Invitrogen) and incubated for 1 hr at RT. Cells were washed in betweenthe treatment steps and treated with a mounting medium containing 2.5%DABCO (Sigma). Stained coverslips were mounted on a clean glass slideusing transparent nail polish and viewed under a Leica confocalmicroscope. The acquired images were analyzed using Leica confocalsoftware. BAL-MNC plated at a concentration of 1×10⁶ per 96-well in aU-bottom plate treated and stained in a similarly also gave comparableresults.

In Vitro Uptake of Nano-KAg by Pig Mφs and Determination of CD80/86Expression by Flow Cytometry

BAL-MNC were seeded in a 24 well plate at a density of 1×10⁶ per ml anduntreated or treated with K-Ag or Nano-KAg (containing a 2, 0.2, and0.02 μg/ml of PRRSV protein) and incubated for 3 hr at 37° C. in a 5%CO₂ incubator. Cells uninfected or infected with PRRSV (MN184 strain) at0.1 MOI for 12 hr at 37° C. in 5% CO₂ incubator served as controls.Cells were treated with anti-PRRSV N′ mAb followed by goat anti-mouseIgG Alexa Flour488, washed and fixed before analysis. To assess theexpression of CD80/86 on professional antigen presenting cells (APCs),BAL-MNC were treated as above for 16 hr at 37° C. in 5% CO₂ incubator,washed and stained using biotinylated human CTLA4-mouse immunoglobulinfusion protein (Ancell, MN) and PE-conjugated CD172 (Southern Biotech)(43), followed by streptavidin percpcy5.5. Cells were fixed using 1%paraformaldehyde and analyzed using FACS Aria II (BD Biosciences) flowcytometer.

Pigs and Inoculations

Conventional large White-Duroc crossbred weaned specific-pathogen-freepigs at 3-4 wks of age were transported to a BSL-2 facility, FAHRP,OARDC. The swine-herd was confirmed seronegative for antibodies toPRRSV, porcine respiratory corona virus, transmissible gastroenteritisvirus, and porcine Circo virus 2. Blood samples collected from pigs onarrival was confirmed negative for PRRSV antibodies. Pigs were allowedto acclimate for an additional week, they received food and water adlibitum and maintained under the supervision of a veterinarian.

In a pre-challenge study, pigs (n=9) were grouped randomly into threegroups (n=3 per group). Group I—mock pigs inoculated with DMEM and PBS;Group II—inoculated with K-Ag; Group III—inoculated with Nano-KAg,inoculated once intranasally. Each vaccine (Nano-KAg and K-Ag) dose hasone mg of crude viral preparation containing ˜5×10⁶ TCID₅₀ ofinactivated virus. All the pigs were euthanized on day post-immunization(DPI) 15 and evaluated for innate and the PRRSV specific immuneresponses. In a post-challenge study, pigs (n=12) were divided randomlyinto four groups (n=3 per group). Group I—mock pigs; Group II—inoculatedwith normal saline; Group III—inoculated with K-Ag; Group IV—inoculatedwith Nano-KAg. Each vaccine dose had same amount of Ags as describedabove. Groups II, III, and IV were challenged with PRRSV MN184 (0.5×10⁶TCID₅₀/ml, 2 ml per pig) on DPI 21 and euthanized on day post-challenge(DPC or PC) 15. All the inoculations were performed once by intranasalroute. Mock-inoculated pigs were separately euthanized beforesacrificing virus challenged animals.

(1) Gross and Histological Analysis

Necropsies were performed on all of the pigs and the lungs and lymphnodes were examined grossly and histologically. Macroscopic pulmonarylesions were given an estimated score based on the percentage ofconsolidated lesions in individual lobes as described previously (44).The lung tissue samples collected from the caudal lobe was fixed in 10%neutral buffered formalin, processed into paraffin blocks, cut intosections (3 μm) and stained using hematoxylin-and-eosin (H&E) asdescribed previously (44). Frozen lung sections (3 μm) wereimmunostained as described previously (45). Briefly, sections weredewaxed, dehydrated, quenched, washed in PBS, blocked using 2% goatserum, and treated with PRRSV nucleocapsid protein specific mAb (SDOW17)(Rural Technologies, Inc.,) or isotype control mAb. The sections weretreated with ABC peroxidase staining kit (Vectastain Elite, Vector Labs)and the labeling was “visualized” by application of DAB(3,3′-diaminobenzidine) substrate (Vector Laboratories) andcounterstained with hematoxylin. Immunostained lung slides were examinedby an unbiased certified veterinary pathologist to score for thepresence of PRRSV Ags.

Collection of Blood and Lung Samples for Analysis

For evaluation of viremia and for titration of PRRSV specific serumneutralizing antibodies, 3 to 5 ml of blood samples were collected onDPI 0 and 21, and DPC 7 and 15. Separated serum was aliquoted and keptat −20° C. Lung homogenates were prepared for cytokine and virusevaluation as described earlier (46, 12).

Isolation of PBMC, Lung MNC, and TBLN Cells

To isolate PBMC, blood was collected in acid citrate dextrose solutionfrom euthanized pigs and processed as described (47). Lung-mononuclearcells (lung MNC/LMNC) from individual pigs were isolated as per thedescribed procedure (48). The airways were lavaged to collect BAL cellsusing sterile ice cold PBS containing EDTA (0.03%) (49). Samples oftracheobronchial lymph nodes (TBLN), iliac lymph nodes (ILN), andtonsils were collected in DMEM, and MNCs were isolated as describedpreviously (48).

Virus Titration and Virus Neutralizing Test (VNT)

PRRSV titer and virus neutralizing antibody titer in serum and in lunglysate was analyzed by indirect immunofluorescence assay (IFA) aspreviously described (37). Briefly, for virus titration confluentmonolayer of MARC-145 cells in 96-well microtiter plate was treated with10-fold dilution of serum for 48 hr. To measure VN titers in serum andlung lysates, samples were heat inactivated, UV treated, twofolddiluted, and incubated with an equal volume of PRRSV (MN184) 250 TCID50per well for 2 hr at 37° C. One hundred microliters of the suspensionwas transferred into 96-well plate containing confluent monolayer ofMARC-145 cells, incubated for 24 hr at 37° C. in a CO2 incubator. Thecells were fixed with 80% acetone and stained with anti-PPRSV N′mAb(SDOW-17) and Alexa-488 conjugated anti-mouse IgG(H+L). Plates weremounted using glycerol-PBS and examined for viral plaques under afluorescent microscope.

PRRSV Specific Isotype Antibody Analysis in the Lungs and Blood

The presence of PRRSV specific IgA and IgG antibodies in serum and lunglysate were analyzed by ELISA. Briefly, ELISA plates were coated withpre-titrated quantity of crude killed PRRSV (MN184) Ags (10 μg/ml) incarbonate-bicarbonate buffer (pH 9.6) overnight at 4° C., washed andtreated with blocking buffer (1% BSA and 0.1% Tween 20 in PBS) for 2 hrat RT; serum (1:100) and lung lysate (0.5 mg/ml, w/v) samples were addedand incubated for 2 hr at RT. The bound virus specific isotype antibodywas detected using anti-pig IgA and IgG secondary antibodies conjugatedwith HRP (KPL). Finally, plates were developed using the chromogen TMBand read at 450 nm. To eliminate the background activity, non-PRRSVspecific antigen-coated control plates were also included, blocked andtreated with test samples side-by-side. The OD values obtained fromexperimental plate were subtracted from the control plate.

PRRSV Specific Recall Cytokine Response

Five million pig PBMC, TBLN MNC, and lung MNC were subjected to ex vivorestimulation in the absence or presence of killed crude PRRSV MN184 Ags(50 μg/ml) as described previously (48), and the harvested supernatantwas analyzed to measure cytokines Cytokines secreted by immune cellscultured in the absence of PRRSV Ags was subtracted from thecorresponding test value.

Analysis of Cytokine Response and Flow Cytometric Analysis of ImmuneCells

Serum samples, harvested culture supernatants, and lung lysates wereanalyzed for Th1 (IFN-γ and IL-12), Th2 (IL-4), pro-inflammatory (IL-6),and immunosuppressive (IL-10 and TGF-β) cytokines by ELISA (48). Amountof cytokines present in the lung lysate was normalized to picogram pergram of lung tissue. Flow cytometry analysis was performed to determinethe phenotype and the frequency of different immune cells by amulticolor immunoassay as described previously (48). Briefly, 50,000events of immunostained cells were acquired using a FACS Aria II (BDBiosciences) flow cytometer and analyzed using FlowJo software (TreeStar, Inc.,). The analysis was done to determine different immune cellpopulations based on the following phenotypes: NK cell rich fraction(CD3−CD4−CD8+); T-helper cells (CD3+CD4+CD8−); CD8+ T cells(CD3+CD4−CD8+]; T-helper/memory cells (CD3+CD4+CD8+); γδ T cells(CD8+TcR1N4+); T-regulatory cells (CD4+CD25+Foxp3+); myeloid cells(CD172+); and dendritic cell rich fraction (CD172+CD11C+SLAII+).

Statistical Analysis

All data were expressed as the mean of three pigs+/−SEM. Statisticalanalyses were performed using one way analysis of variance (ANOVA)followed by post-hoc Tukey's test using GraphPad InStat (softwareversion 5.0) to establish differences between K-Ag and Nano-KAg piggroups. Statistical significance was assessed as P<0.05.

Results

In Vitro Characterization of PRRSV Entrapped-Nanoparticles

Morphology of sham and PRRSV Ags entrapped PLGA nanoparticles wasdetermined by scanning electron microscopy (SEM) which revealed the sizeof particles to be 200-600 nm (FIG. 7A). The average protein content innanoparticles or core-loading was 0.50-0.55% (w/w), which represents anencapsulation efficiency of 50-55%. Upon re-dispersion of the Nano-KAgin PBS, PRRSV proteins were released slowly in the first 48 hr, later agradual release profile was observed over the next 5-weeks.

Uptake of Nano-KAg by APCs was studied using BAL-MNC harvested fromthree healthy SPF pigs. The confocal images revealed preferential uptakeof Nano-KAg by APCs but not unentrapped viral Ags (K-Ag), and PRRSVinfected cells served as a positive control (FIG. 7B). Engulfednanoparticles delivered the PRRSV Ags to early endosomes and it wascomparable to virus-infected control (FIGS. 7C & D). Further, Nano-KAgengulfed APCs underwent maturation as indicated by significantlyincreased expression of CD80/86 on APCs compared to K-Ag treated cells(FIG. 7E). In addition, 57% BAL-MNC treated with Nano-KAg were positivefor PRRSV protein comparable to virus infected cells (FIG. 7F ii & iv).In contrast, only 9% BAL-MNC treated with K-Ag were positive for viralprotein (FIG. 7F iii). The results show that PRRSV Ags delivered innanoparticles were phagocytosed by APCs and the released protein wasfound inside the endosomes.

Potential of PRRSV Ags Entrapped Nanoparticle (Nano-KAg) as a CandidateVaccine

In a pre-challenge study, intranasal delivery of Nano-KAg resulted ininduction of innate immune response at both mucosal and systemic sites,indicated by a significant increase in the frequency of NK cells, DCs,and γδ T cells. In addition, immune cells involved in adaptive arm ofthe immunity, such as Th/memory cells and CD8+ T cells were increasedsignificantly in the lungs of Nano-KAg compared to K-Ag immunized pigs(FIG. 8 A-E). Similarly, PBMC of Nano-KAg vaccinated pigs showed asignificant increase in the frequency of γδ T cells and DCs (FIGS. 8 H &I). Interestingly, K-Ag vaccinated pigs had a reduction in the frequencyof NK cells and Th/memory cells in the lungs (FIGS. 8 A & D). Further,lung MNC and PBMC from Nano-KAg immunized pigs secreted significantlyreduced levels of the cytokine IL-10, and higher amounts of IL-6 in arecall response (FIG. 8 F, G, J). In addition, innate cytokine IFN-α wassecreted in pigs immunized with Nano-KAg and not K-Ag (FIG. 8K).

Significant Reduction in the Lung Pathology and Virus Load in Nano-KAgVaccinated Pigs

Initially, dose-dependent response to the Nano-KAg candidate vaccine wasperformed in vivo in pigs using doses of 0.2 and 1 mg per pig (˜1×10⁶and 5×10⁶ TCID50 of inactivated virus), and detected appreciable immuneresponse with one mg dose. Therefore, all the subsequent trials wereperformed with a dose of one mg per pig. In a post-challenge study,Nano-KAg immunized MN184 challenged pigs were clinically healthy with nofever or respiratory distress. In contrast, both K-Ag and unvaccinated,MN184 virus challenged pigs had fever with reduced feed intake duringthe first week post-challenge. During necropsy, significantly reducedgross lung lesions were observed in the Nano-KAg immunized groupcompared to other two virus challenged groups (FIG. 9C). Microscopicexamination of H&E stained lung sections of unvaccinated and K-Agvaccinated, MN184 challenged pigs had severe pneumonic lesions withmassive infiltration of mononuclear cells with large infected area. Incontrast, significantly reduced lung lesions were observed in Nano-KAgimmunized virus challenged pigs (FIG. 9A).

Immunohistochemistry analysis has revealed abundant inactivated PRRSVpositive cells in the lung sections of unvaccinated and K-Ag immunized,MN184 virus challenged pigs compared to Nano-KAg pig group (FIGS. 10B&D). PRRSV virus titer in serum samples and lung homogenate hasindicated reduced viral load by greater than one log at daypost-challenge (DPC or PC) 7 with complete viral clearance by DPC 15 inNano-KAg vaccinated pigs compared to K-Ag vaccinated and unvaccinatedpigs (FIG. 9E). The K-Ag vaccinated pigs also cleared the viremia betterthan the unvaccinated pigs (FIG. 9E). Similarly, PRRSV load in the lungswas also reduced in Nano-KAg compared to K-Ag immunized, MN184 viruschallenged pigs (FIG. 9F). Also the PRRSV titer values (TCID50/ml) inboth the serum and lung homogenate showed a reduction in Nano-KAgimmunized compared to control pigs.

Humoral Immune Response in Serum, Lungs and Nasal Swab

Lung homogenates of Nano-KAg immunized pigs contained significantlyhigher levels of secreted virus specific IgA and IgG antibodies comparedto unvaccinated or K-Ag immunized, MN184 challenged pigs (FIGS. 10 A&B).In the serum samples of Nano-KAg immunized pigs increased IgA antibodylevels at PC 0, with a significant increase at PC 15 compared to eitherunvaccinated or K-Ag immunized, MN184 challenged pigs was detected (FIG.10D). The PRRSV specific IgG antibody levels in serum (FIG. 10E), andboth IgA and IgG levels in the nasal swab (FIGS. 10G&H), weresignificantly higher in Nano-KAg immunized compared to K-Ag immunizedpigs at DPC 15. Although increased PRRSV specific neutralizing antibody(VN) titers in the lungs and serum were detected at DPC 15, the data wasnot statistically significant (FIGS. 10 C&F).

Nanoparticle-Based PRRSV Vaccine Showed Enhanced Innate Immune Responsein Pigs

Nano-KAg vaccine received MN184 virus challenged pigs had significantlyincreased innate IFN-α production in the lungs (FIG. 11A). In K-Agimmunized virus challenged pigs a fourfold reduction in NK cellfrequency compared to mock pigs was detected; in contrast, in Nano-KAgvaccine inoculated pigs the NK cell population was comparable to mockpigs (FIG. 11B). Further, lung NK cell-cytotoxic function inunvaccinated and K-Ag immunized, MN184 virus challenged pigs wascompletely suppressed; however, in Nano-KAg received pigs it waspartially decreased (FIG. 11C). The frequency of γδ T cells and CD4+(butnot CD8+) T cells in the lungs of Nano-KAg vaccinated animals weresignificantly increased compared to K-Ag and unvaccinated, viruschallenged pigs (FIGS. 11 DE&F). In the peripheral blood of Nano-KAgimmunized pigs a significantly increased frequency of DCs, and in TBLNsignificantly increased frequency of both DCs and γδ T cells wasobserved (Table 1).

Table 1 shows pigs that were unvaccinated or vaccinated with either K-Agor Nano-KAg once intranasally and challenged with PRRSV strain MN184 andeuthanized on DPC 15. Different immune cell subsets present in PBMC andTBLN MNC were enumerated by flow cytometry. ^(a) CD172⁺ cells were gatedto enumerate CD11c and SLAII expression and the percent of DCs richfraction (CD172⁺CD11c⁺SLAII⁺) is shown. ^(b) CD3⁺ cells were gated toenumerate CD4 and CD8α expression and the percent of NK cell richfraction (CD3⁻CD4⁻ CD8α⁺) is shown. ^(c) CD25⁺ cells were gated toenumerate CD4 and Foxp3 expression and the percent of CD4⁺CD25⁺Foxp3⁺cell is shown. Each number is an average percent of immune cells fromthree pigs+/−SEM. Asterisk represents the statistical significantdifference (p<0.05) between Nano-KAg and K-Ag received pig groups.

TABLE 1 Frequency of immune cells in PBMC and TBLN of Nano-KAgvaccinated pigs. Immune Unvac- cells Mock cinated K-Ag Nano- PBMCMyeloid cells 21.1 ± 0.5  59.3 ± 4.3  69.9 ± 3.7  72.8 ± 2.5  DendriticCells 3.1 ± 0.6 0.01 ± 0.3  0.7 ± 0.1 3.03 ± 0.6* γδ T cells 2.2 ± 0.51.1 ± 0.1 2.7 ± 0.3 4.7 ± 0.7 NK cells ^(b) 4.1 ± 0.3 32.6 ± 10.0 13.2 ±1.6  16.4 ± 1.6  Tregs ^(c) 0.3 ± 0.1 0.9 ± 0.2  1.8 ± 0.1* 0.9 ± 0.1TBLN MNC Myeloid cells 1.7 ± 0.8 18.2 ± 0.6  15.9 ± 3.6  18.6 ± 5.2 Dendritic Cells 1.5 ± 0.0 0.8 ± 0.1 0.1 ± 0   5.6 ± 1.2* γδ T cells 1.8± 0.1 1.1 ± 0.4 1.5 ± 0.3  8.6 ± 0.9* NK cells 5.1 ± 0.6 3.7 ± 0.3 1.3 ±0.2 8.5 ± 3.7 Tregs 1.3 ± 0.7 3.1 ± 0.2 2.1 ± .02  1.8 ± 0.04Suppression of Immunosuppressive Cytokine but Boosting of IFN-γ Responseby Nano-KAg

Pigs immunized with Nano-KAg vaccine showed significantly reduced Tregpopulation in the lungs comparable to mock pigs (FIG. 12A). In contrast,unvaccinated and K-Ag immunized MN184 virus challenged pigs had higherlevels of Treg cells compared to Nano-KAg group (FIG. 12A).Immunosuppressive cytokine response was strongly suppressed in Nano-KAgvaccinated pigs, indicated by significantly reduced levels of IL-10 andTGF-β in the lung homogenate; and also their increased secretion in arecall response of lung MNC was observed in Nano-KAg vaccinated comparedto both unvaccinated and K-Ag immunized virus challenged pigs (FIGS. 12B, C, E, & F). In contrast, a significantly increased IFN-γ in lunghomogenate and its secretion in a recall response of lung MNC wasdetected in Nano-KAg vaccinated compared to control pigs (FIGS. 12 D&G).

Cultured restimulated immune cells of Nano-KAg immunized pigs secretedsignificantly reduced IL-10 (both PBMC and TBLN-MNC) and TGF-β (PBMC),and an increased secretion of IFN-γ (both PBMC and TBLN-MNC) compared tounvaccinated and K-Ag immunized MN184 challenged pigs (FIGS. 13 A, B, C,E, & F). The level of proinflammatory cytokine, IL-6, in a recallresponse was significantly reduced in both PBMC and TBLN-MNC in Nano-KAgcompared K-Ag immunized virus challenged pigs (FIGS. 13 D&G).

As disclosed herein, a rapid uptake of Nano-KAg vaccine by lung APCsfollowed by translocation of viral Ags into endosomal compartment, andincreased expression of the activation marker CD80/86 on APCs wereobserved. These data show that virus specific adaptive immune responsecould be elicited in the respiratory tract of pigs using PLGAnanoparticle-based killed PRRSV vaccine. Differential cell counts fromBAL fluid harvested from healthy mice, humans, and pigs have indicatedthat greater than 90% of cells are alveolar macrophages (62; 63; 64),showing that intranasally delivered Nano-KAg are phagocytosed byalveolar macrophages which serve as principle APCs in the lungs.

The pre-challenge study disclosed herein has demonstrated the capabilityof intranasally delivered candidate Nano-KAg vaccine to significantlyincrease the frequency of CD8+ T cells, Th/memory cells, withconcomitant increase in the secretion of innate (IFN-β), proinflammatory(IL-6), and Th1 (IFN-γ) cytokines.

The post-challenge study using a virulent heterologous PRRSV has provedthe ability of Nano-KAg vaccine to induce better cross-protectiveimmunity in pigs. Immunologically, PRRSV modulates the innate immunefunction of pigs by dampening the IFN-α production, reduces the NK cellpopulation and its cytotoxic function, which lead to weak/delayedadaptive immune response (28, 57). However, pigs vaccinated intranasallywith Nano-KAg prevented a majority of virus induced immunosuppressivemechanisms with concomitant boost in innate and virus specific adaptiveimmune responses. γδ T cell is an important innate immune cell atmucosal sites and they possess non-MHC class I cytolytic activity. InNano-KAg immunized pigs, increased population of γδ T cells, in additionto NK cells, CD4+ and CD8+ T cells, and increased secretion of IFN-γwere detected at both mucosal and systemic sites.

PRRSV infected pigs can be immunosuppressed due to increase in thepopulation of Tregs and secretion of immunoregulatory cytokines, IL-10and TGF-β (28, 75), and inhibition of IFN-γ production in pigs (77, 109,78). Nano-KAg immunized pig lungs, TBLN and blood had significantlyreduced Tregs associated with decreased IL-10 and TGF-β and increasedIFN-γ secretion compared to unvaccinated and K-Ag vaccinated viruschallenged pigs. In a pre-challenge study, increased secretion ofproinflammatory cytokine IL-6 in Nano-KAg immunized pigs appears to beinvolved in initiation of adaptive immune response, and its diminishedproduction in immunized post-challenged pigs was associated with reducedinflammatory lung pathology.

In Nano-KAg immunized pigs increased levels of PRRSV specific IgA, IgG,and VN antibody titers in the lungs, blood, and nasal wash wereobserved. Mucosal immunization can induce the production of IgAantibodies and effector response at distant tissues (21). The IgAantibody is protective against various viral infections and they possesssignificant virus neutralization activity at both mucosal surfaces andblood (50-53).

An important discovery to swine farmers and researchers is that the pigsimmunized with Nano-KAg vaccine cleared the viremia of a virulentheterologous PRRSV in two weeks. Microscopic lung study in Nano-KAgimmunized pigs was associated with the protective immune correlates andreduced viral load in the lungs.

Example 3 Nanoparticle-Based Adjuvanted Inactivated Porcine Reproductiveand Respiratory Syndrome Virus Vaccine Elicits Superior Cross-ProtectiveImmunity in Pigs

Porcine reproductive and respiratory syndrome (PRRS) is responsible forgreater than $664 million direct annual loss to the US pork industry(8). The causative agent, PRRS virus (PRRSV), is an enveloped positivestrand RNA virus belongs to the family Arteriviridae. There are twoknown PRRSV genotypes, European (type I) and the North American (typeII), with varying inter and intra genotypic genetic and antigenicdiversity, signifying extreme mutagenic nature of PRRSV (38).

For about the last 20 years both modified live (PRRS-MLV) andinactivated PRRSV vaccines have been in use, but still control of PRRSremained unsuccessful. Moreover, PRRS-MLV has been implicated in thespread of mutated vaccine virus to susceptible pigs (32). Availableinactivated vaccines are safe but poorly immunogenic (117). Therefore,development of a potent inactivated PRRSV vaccine is warranted. PRRSVprimarily infects pigs through respiratory and genital mucosa, and itsprimary targets are macrophages (118). Intranasal delivery of a vaccineis non-invasive and has the ability to stimulate a protective immuneresponse not only at the respiratory tract, but also in the genitaltract and systemically (119). UV-irradiation or BEI inactivation ofPRRSV preserves the functional immunogenic epitopes (100). Efficient Th1response-inducing adjuvants are necessary in subunit/inactivatedvaccines (120). Recently, the potent adjuvanticity of M. tb whole celllysate (WCL) to PRRS-MLV was identified (28).

Biodegradable PLGA (poly lactide-co-glycolide) based micro/nanoparticlesare widely used for targeted/sustained delivery of drugs and vaccines(121). PLGA is an FDA and European Medicines Agency (EMEA) approvedagent (92). Intranasally delivered particulate antigens are sampled byM-cells of nasal lymphoid tissues, which in turn deliver to underlyingprofessional antigen presenting cells (APCs) in the respiratory tract(122). Recently, it has been shown that a single dose of PLGAnanoparticle (NP) based inactivated PRRSV (NP-KAg) vaccine deliveredintranasally to pigs elicits an immune response with substantial viralclearance (123). Coadministration of NP-KAg vaccine twice withnanoparticle entrapped/unentrapped M. tb WCL, intranasally, can elicitrobust anti-PRRSV cross-protective immunity with complete viralclearance in pigs. These results demonstrate that NP-KAg vaccinecoadministered with unentrapped M. tb WCL completely cleared theinfective heterologous PRRSV both from the lungs and systemically.

Materials and Methods

Reagents

MARC 145 cells (14) were used to prepare PRRSV stocks and assays. Cellswere maintained in DMEM with 10% FBS. For virus infection, DMEM with 2%horse serum was used. North American prototype PRRSV strain VR2332 (15)was used in vaccine preparation, and PRRSV MN184 (15). Mycobacteriumtuberculosis whole cell lysate (M. tb WCL) was prepared as previouslydescribed (21).

Preparation of Vaccine Antigens and PLGA Nanoparticle-Based VaccineFormulations

PRRSV strain VR2332 (38) antigens was grown and UV-killed (KAg) asdescribed previously to use in the vaccine (123). PLGA NPs entrappedwith KAg (NP-KAg) or M. tb WCL (NP-M. tb WCL) were prepared by doubleemulsion method (w/o/w) as described (123, 41).

Determination of Protein Entrapment Efficiency and Characterization ofNP-KAg

Entrapped protein in NPs was estimated as described (123). Morphology ofthe Nano-KAg was visualized using the Philips XL30-FEG scanning electronmicroscope (SEM) at 20 kV at 30,000× magnification. Size distribution ofthe sham or KAg entrapped NPs was measured using NICOMP 370 particlesizer (Particle Sizing Systems, CA). The zeta potential of the NPs wasdetermined by ZetaPALS (Brookhaven Instruments Corp., NY).

Determination of In Vitro Protein Release from NP-KAg Vaccine

The assay was performed as previously described (124). Briefly, 50 mgNP-KAg was suspended in one ml PBS and the supernatant was collectedimmediately to estimate the burst release. The pellet of NP-KAg wasresuspended repeatedly with one ml PBS and the supernatants werecollected at 1, 5, 10, 15, 20, 25, and 30 days and stored at −20° C. Onday 30, undegraded NPs were lysed to recover the protein, and all thesamples were estimated for protein concentration by BCA method.

In Vitro Uptake of Nano-KAg by PAM Cells

Nano-KAg vaccine containing 2 μg of PRRSV KAg were suspended in one mlof RPMI and incubated with PAM cells (3D4/2, ATCC) for 0, 5, 20, 30 min,3, 12, and 24 hr. KAg (2 μg), sham NPs, and cells infected for 24 hrwith PRRSV VR2332 strain (MOI=1) were included as controls. Cells werewashed and fixed with acetone and incubated with PRRSV N′ mAb SDOW17(Rural Technologies, Inc., SD) followed by anti-mouse IgG (H+L)Alexa-488 (Life Technologies, Grand Island, N.Y.), and observed underthe inverted fluorescent microscope.

Pigs and Inoculations

Conventional Large White-Duroc crossbred, specific-pathogen-free weaned(3-4 weeks) piglets were procured from a swineherd seronegative forPRRSV, PRCV, TGV, and PCV2 antibodies. A total of 30 pigs were randomlydivided into one of the 10 groups (3 pigs/group), and indicated vaccineformulation was inoculated (2 ml) twice at two-week interval (Table 2).Except pigs in group 1 all the other groups were challenged onpost-vaccination day 28 with a virulent heterologous North AmericanPRRSV (type II) strain MN184 (5×10⁵ TCID₅₀/pig) (38). Adjuvant andvaccine were entrapped separately and combined before administering themintranasally. The doses of the adjuvant and NP-KAg were tested earlier(123, 95). Pigs were euthanized on post-challenge day (PC) 15. Animalswere maintained with food and water ad libitum, samples collected andeuthanized as per the approved protocol of Institutional Animal Care anduse Committee, The Ohio state University.

Table 2 shows a total of 30 pigs (N=30) were divided in ten groups eachhaving three pigs (n=3). Groups 1 and 2 served as mock and mockvaccinated-challenged respectively. Other 8 groups were divided in totwo dose categories of either 100 or 500 μg/pig of vaccine dose asdetailed above. For statistical comparison, groups 1 and 2 wereconsidered with both the categories.

TABLE 2 Pig groups used in the experiment. GP. No. Experimental groups 1Mock pigs (PBS and DMEM) 2 Mock + Challenged with PRRSV- MN184 strainVaccine Dose (100 μg/pig) + Vaccine Dose (500 μg/pig) + Chal. Chal. 3100 μg of K-Ag 3. 500 μg of K-Ag 4 100 μg of K-Ag + 4. 500 μg K-Ag/pig +M. tb WCL M. tb WCL 5 100 μg of NP-KAg + 5. 500 μg of NP-K-Ag + NP-M tbWCL NP-M. tb WCL 6 100 μg of NP- K-Ag + 6. 500 μg of NP-K-Ag + M. tb WCLM. tb WCLCollection of Blood and Lung Samples for Analysis

Maximum PRRSV load in blood, lymphoid tissues, and lungs are detected atdays 6 to 15 post121challenge/infection (125). Therefore, heparinizedblood samples (3-5 ml) were collected on the day of vaccination and atPC 0, 6, 10, and 15, and plasma was aliquoted and preserved at −70° C.Pigs were monitored daily for the respiratory disease and rectaltemperature, and the body weights were recorded twice weekly.

Preparation of Lung Homogenate and Isolation of MNCs from the Lungs andBlood

Lung homogenate was prepared as described (19). The lungs were lavagedwith cold PBS containing antibiotics and bronchoalveolar lavage (BAL)fluid was recovered, clarified, aliquoted, and stored at −70° C. Bloodwas collected in ACD solution and peripheral blood mononuclear cells(PBMC) isolated (20), and from lung tissue samples lung MNCs (LMNC) wereisolated (52).

Analysis of PRRSV Specific Antibodies

The assay was performed as described previously (53). Briefly, ELISAplates were coated with pre-titrated MN184 Ags or with PRRSV recombinantN, M, or GP5 proteins in carbonate buffer. For plasma mean OD value of10-fold dilution of 30 PRRSV negative samples plus 2 times standarddeviation (SD) was considered as the positive-negative cut-off.Reciprocal of the highest dilution of the plasma sample which had the ODvalue above the cut-off OD was considered as the end titer. PRRSVspecific IgG1 and IgG2 antibodies in the lungs and plasma samples wereanalyzed as described (54).

PRRSV Specific IFN-γ Secreting Cells (ISCs) and Estimation of Cytokines

ELISPOT assay was performed to analyze ISCs as described (55). In therecall cytokine response, the supernatant harvested from PBMC culturesrestimulated with killed MN184 Ags was analyzed for IL-4, IL-6 and IL-10by ELISA as described (56). Lung homogenates were analyzed for IFN-γ,IL-12, IL-6, IL-10, and TGF-β by ELISA (21).

Avidity of the PRRSV Specific Antibodies in the Lungs and Blood

The assay was performed as described (57) with a few modifications.Briefly, to PRRSV Ags coated plate, test samples 1:100 diluted plasma,1:10 lung homogenate, or undiluted BAL fluid was added and incubated;subsequently, serial two-fold diluted NH₄CN (5M to 0.313M) was added.The OD value of the test samples in NH₄CN untreated (0 M) wells wasconsidered as 100%, and the test samples OD value in NH₄CN treated wellswas calculated to reveal the percent retained Ag-Ab complex, expressedin percent.

Hematoxylin and Eosin Staining of Lung Tissue

The lung tissue samples collected from the right cranial lobe was fixedin 10% neutral buffered formalin and processed into paraffin blocks.Five micron sections were cut and stained for hematoxylin-and-eosin(H&E) as described previously (58).

Virus Titration and Virus Neutralization Assays

PRRSV titer and virus neutralizing antibody (VN) titer in lunghomogenates and plasma samples were analyzed by the indirectimmunofluorescence assay (37, 48). For VN assay, serially dilutedsamples were incubated with one of the PRRSV s 128 trains, MN184 (250TCID₅₀), PRRSV 1-4-4 (accession #10-16734) (126) (100 TCID₅₀), orSD03-15 (21) (200 TCID₅₀). The PRRS viral titers in each gram of lungtissue or per ml of plasma, and virus specific VN titers weredetermined.

Determination of PRRSV RNA

Viral RNA copy number was detected in the lung homogenate (per gm oflung tissue) and BAL fluid (per ml) by quantitative real-time PCR asdescribed (128).

Statistical Analysis

The data were expressed as the mean±SEM of three pigs. Statisticalanalyses were performed by one way ANOVA followed by Tukey's t-testusing GraphPad Instat3 software. Comparisons were performed betweendifferent treatment groups as mentioned below, and p<0.05 was consideredfor statistical significance (alphabets a to j).

-   a→Group 2 vs Group 3, b→Group 2 vs Group 4, c→Group 2 vs Group 5-   d→Group 2 vs Group 6, e→Group 3 vs Group 4, f→Group 3 vs Group 5-   g→Group 3 vs Group 6, h→Group 4 vs Group 5, i→Group 4 vs Group 6,    and-   j→Group 5 vs Group 6    Results    NP-KAg Vaccine was Rapidly Endocytosed by Porcine Alveolar    Macrophages (PAMs) and Sustained Release of Entrapped Ags Over    Several Weeks

Potency of NP mediated delivery of drugs or vaccines depends on theirloading capacity and size (129). The entrapment efficiency ofkilled-PRRSV (KAg) and M. tb WCL in NPs was about 50-60%. Sham orentrapped NP particles were circular with smooth surface (FIG. 24A).Dynamic light scattering (DLS) of NPs measured their diameter baseddistribution, and the mean diameter±SD of sham, NP-KAg, and 151 NP-M. tbWCL were 480±53, 520±41, and 650±98 nm, respectively. Further, 85% ofsham NPs, 92% of NP—M. tb WCL, and 78% of NP-KAg were in the size rangeof 400-700 nm. Interestingly, there was no difference among all threeNPs with respect to surface electrostatic potential (−26 mV) as measuredby Zeta potential, indicating that differential surface charge ofentrapped proteins did not influence the net electrostatic potential offinally formed NPs.

In Vitro Protein Release Profile of Nanoparticle Entrapped InactivatedPRRSV (NP-KAg)

The surface associated protein in nanoparticles is equivalent to theamount of protein released at time zero (i.e., immediately afterreconstitution in PBS), called as burst release (130); and it was 9.5%in NP-KAg vaccine. The release during first 24 hr after reconstitutionwas 30.5% (FIG. 24B), and after 30 days 61% of the entrapped protein wasreleased (FIG. 24B). The remaining 39% of viral Ags was recovered in theun-lysed NPs. Even one year old NP-KAg stored at −20° C. had 13.6% burstrelease, and 76% of released antigens by 30 days, indicating that PLGANPs retain the entrapped vaccine beyond one year and allow its sustainedrelease over a period of several weeks under normal physiologicalconditions (FIG. 24B). Thus, the results indicated that as expected PLGAnanoparticles could efficiently retain and permit sustained release ofentrapped PRRSV Ags over a long period of time.

In Vitro Uptake of NP-KAg Vaccine by PAM Cells

PRRSV is host-specific, infects only pigs and the virus infectspulmonary alveolar macrophages (PAM). Treatment of PAM cells with thevaccine preparations revealed very little uptake of unentrapped KAg over24 hr (FIG. 24Ci). In contrast, a rapid uptake of NP-KAg as early as 5min and reached the peak by 30 min, followed by a gradual reduction inthe fluorescence signals after 3 hr post-treatment (FIG. 24Cii);possibly due to degradation of Ags. Control sham nanoparticles treatedand untreated PAM cells did not show any fluorescence signals (FIG.24Ciii & Civ), while the PRRSV infected cells had virus specific greensignals (FIG. 24Cv).

Enhanced Production of PRRSV Specific IgA in the Airway Surface and BothIgA and IgG in the Lung Parenchyma in Adjuvanted NP-KAg Immunized Pigs

PRRSV specific IgA response was significantly higher in the BAL fluidand lung homogenate in group 6 pigs at PC 15 (both with 100 and 500 μgvaccine doses) compared to other test groups (FIGS. 25A & C). Incontrast, comparable levels of virus specific IgG were detected in theBAL fluid of all the vaccine trial groups (FIG. 25B). In the lunghomogenate a significantly higher levels of virus specific IgG wereobserved in pig groups 4 and 6 (FIG. 25D). The results indicated that inadjuvanted NP-KAg vaccinated pigs, virus specific IgA is a majorantibody isotype in alveolar surfaces, while both IgA and IgG in thelung parenchyma. In the plasma of group 6 pigs (both vaccine doses), IgGresponse was significantly higher compared to group 2 pigs from PC 0 to15 (FIGS. 25E & F). Overall, significantly increased levels of PRRSVspecific antibodies were produced in NP-KAg+M. tb WCL vaccinated pigs.Furthermore, PRRSV specific IgG response against recombinant structuralproteins was evaluated and comparable levels of IgG titers against viralsurface glycoprotein GP5 and M protein were found (FIGS. 30 A, B, C &D). While PRRSV nucleocapsid (N) protein specific IgG antibodies weresignificantly high in group 6 pigs (FIGS. 30E & F). These resultsindicated that NP-KAg+M. tb WCL induced a strong humoral immuneresponses.

Broadly Cross Protective PRRSV Neutralization Response Elicited byAdjuvanted NP-KAg

Neutralizing antibodies play an important role in the clearance of PRRSVinfection (131). In group 6 pigs, the virus neutralization (VN) titersin the lungs against MN184 strain was significantly higher with meantiters 16 and 27 in pigs received 100 and 500 μg vaccine doses,respectively, compared to other groups (FIGS. 26 A & B). Plasma of group6 pigs (at both the vaccine doses) had significantly higher VN titerscompared to other groups (FIGS. 26C & D). Compared to 100 μg vaccinedose, the 500 μg dose elicited a steady increase in plasma VN titers ingroup 6 pigs (FIG. 26D). Against another antigenically divergent type IIPRRSV strain 1-4-4 and a type I PRRSV strain SD03-15 (both these strainsare genetically highly divergent from VR2332 and MN184 strains),significantly increased VN titers were detected in the lung homogenateof group 6 pigs (only with 500 μg dose) compared to group 5 and 3,respectively (FIGS. 26F & H). While the VN titers against PRRSV 1-4-4and SD03-15 strains remained undetectable in the plasma until PC 10; andonly in group 6 pigs (500 μg dose) a VN titer of eight against SD03-15was detected at PC 15. These results indicated that adjuvanted NP-KAgelicits could elicit cross-reactive VN response.

Downregulated Immunosuppressive and Enhanced (and Balanced) Th1-Th2Cytokine Response in Adjuvanted NP-KAg Inoculated Pigs

Group 6 pigs (both the vaccine doses) had significantly increasedinterferon-γ secreting cells (ISCs) in lung mononuclear cells (LMNC)compared to groups 3, 4, and 5 (FIGS. 27A & 33A). The IFN-γ productionin the lung homogenate at PC 15 was significantly increased in group 6pigs compared to groups 4 and 5 in 500 μg dose category (FIG. 27B), andcompared to only group 5 in 100 μg dose category (FIG. 33B). Productionof another important Th1 cytokine, IL-12, was significantly increased ingroups 4 and 220 6 pigs (500 μg dose) (FIG. 27D). Production of aproinflammatory cytokine IL-6 in the lungs was significantly reduced inall the vaccine trial groups compared to group 2 (FIGS. 27C & 33C). Thecytokines TGF-β and IL-10 are considered as immunoregulatory andimmunosuppressive in nature. Production of TGF-β was significantlyreduced in the lungs of group 6 pigs compared to group 2 (FIGS. 27E &33E), and the levels of IL-10 were comparable in all the tested groupsat PC 15 (FIGS. 27F & 33F). Secretion of IL-6 by restimulated PBMC ofgroup 6 pigs was significantly reduced compared to group 2 (500 μg dose)(FIG. 27H). Interestingly, secretion of IL-10 by PBMC in a recallresponse was significantly reduced in group 6 pigs compared to groups 2and 3 in both the vaccine dose category (FIGS. 27I and 33I). Thecytokine IL-4 is an important indicator of a Th2 response, andsignificantly increased secretion of IL-4 by restimulated PBMC wasobserved in group 6 pigs compared to all other groups which received 500μg dose of vaccine (FIG. 27G). These data clearly indicated thatadjuvanted NP-KAg induced increased Th1 and Th2 and reducedimmunosuppressive cytokines production, which was correlated with theproduction of balanced Th1-Th2 antibody sub-isotypes (FIGS. 32A & B).

Enhanced Frequency of IFN-γ Secreting Lymphocytes and APCs in AdjuvantedNP-KAg Immunized Pigs

LMNC and PBMC were immunostained and analyzed for lymphoid and myeloidcells, and IFN-γ⁺ cells. Groups 6 pigs showed significantly higherpopulation of total IFN-γ producing cells both in LMNC and PBMC (FIGS.28A & J and 34A & J). Pig groups 3, 4, and 6 (500 μg dose) hadsignificantly increased activated CD4 cells (CD4⁺CD8⁻CD25⁺241) in LMNC(Table 3, B), but significantly increased IFN-γ⁺CD4⁺ cells only in group6 pigs in both LMNC and PBMC (FIGS. 28B & K). IFN-γ⁺CD8⁺ cells weresignificantly increased in group 6 pigs in both LMNC and PBMC (FIGS. 28C& L). In the lungs, IFN-γ⁺CD4⁺CD8⁺ cells were significantly higher ingroup 6 pigs than all other tested groups (FIG. 28D), and compared togroups 3 and 5 in PBMC (FIG. 28M). Increased frequency of activated γδ Tcells in group 6 pigs was detected in group 6 pigs compared to othergroups (Table 3, A & B). Both in LMNC and PBMC a significantly increasedIFN-γ⁺ γδ T cell population in group 6 pigs were detected (FIGS. 28E &N). Although there was no significant difference in NK (CD56+) cellfrequency (FIGS. 28F & O), a significant increase in IFN-γ+ 250 NK cellfrequency was detected in group 6 pigs compared to other tested groups(FIGS. 28G & P). With regards to myeloid cells in LMNC, significantlyincreased SLAII⁺ macrophages (MΦs) (CD172⁺CD163⁺SLAII⁺) in group 6 pigscompared to group 5 (FIG. 28H), but not in PBMC were detected (FIG.28Q). In addition, dendritic cells (DCs) rich population(CD172+CD11c+SLAII+) was significantly higher in group 6 pigs comparedto group 5 in LMNC (FIG. 28I), and compared to all other tested groupsin PBMC (FIG. 28R). A similar trend (but not significant in some groups)in lymphoid and myeloid cell frequencies in 100 μg/pig dose receivedgroup 6 pigs was observed (FIG. 34 and Table 3, A).

Table 3 shows the frequency of indicated activated (CD25⁺) lymphocytesubsets present on the day of euthanization in the LMNC (A) and PBMC (B)of pigs vaccinated or unvaccinated with indicated vaccine and adjuvantcombination and challenged with PRRSV MN184. Each number is an averagefrequency of indicated immune cell from three pigs±SEM. Lower casealphabet indicates statistically significant (p<0.05) difference betweentwo indicated groups of pigs as described in materials and methods.

TABLE 3 The populations of activated lymphocyte subsets. KAg + NP (KAg +NP-KAg + Mock Mock + Ch KAg + Ch. WCL + Ch. WCL) + Ch WCL + Ch. (1) (2)(3) (4) (5) (6) (A) 100 μg/pig vaccine dose category LMNC CD4⁺CD8⁻CD25⁺7.2 ± 0.7 7.3 ± 1.2 19.9 ± 9.2  16.3 ± 1.2  3.3 ± 1.1 20.7 ± 2.2 CD4⁻CD8⁺CD25⁺ 4.5 ± 0.4 4.2 ± 0.4 0.3 ± 0  0.5 ± 0.2 0.2 ± 0.1  2.1 ±0.7^(gij) γδ⁺CD25⁺ 0.1 ± 0  0.4 ± 0.3 0.5 ± 0.3 0.7 ± 0.3 0.2 ± 0    1.9± 0.3^(dgij) PBMC CD4⁺CD8⁻CD25⁺ 5.0 ± 1.3 7.7 ± 1.7 10.7 ± 6.2  17.8 ±3.0  4.41 ± 1.62 12.2 ± 1.0  CD4⁻CD8⁺CD25⁺ 6.1 ± 2.8 6.9 ± 1.1 2.6 ± 1.43.3 ± 0.2 1.68 ± 1.19 5.2 ± 2.1 γδ⁺CD25⁺ 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.11.0 ± 0.5 0.54 ± 0.16 0.8 ± 0.2 (B) 500 μg/pig vaccine dose categoryLMNC CD4⁺CD8⁻CD25⁺ 7.2 ± 0.7 7.3 ± 1.2 17.0 ± 1.5^(af)  16.2 ± 1.1^(bh)3.3 ± 1.2 10.8 ± 2.2^(j)  CD4⁻CD8⁺CD25⁺ 4.5 ± 0.4 4.2 ± 0.4 1.2 ± 0.60.9 ± 0.4 0.1 ± 0  1.3 ± 0.3 γδ⁺CD25⁺ 0.1 ± 0  0.4 ± 0.3 1.4 ± 0.5 0.8 ±0  0.3 ± 0   2.3 ± 0.2^(dij) PBMC CD4⁺CD8⁻CD25⁺ 5.0 ± 1.3 7.7 ± 1.7 13.3± 6.2  19.3 ± 7.4  5.7 ± 0.7 31.3 ± 13.6 CD4⁻CD8⁺CD25⁺ 6.1 ± 2.8 6.9 ±1.1 4.1 ± 0.7 1.8 ± 1.0 1.6 ± 0.4 7.3 ± 3.4 γδ⁺CD25⁺ 0.2 ± 0.1 0.3 ± 0.10.2 ± 0.1 0.8 ± 0.3 1.3 ± 0.3  2.3 ± 0.8^(dg)Reduced Lung Pathology, Complete Clearance of Replicating PRRSV, andReduced Viral RNA Load in Adjuvanted NP-KAg Vaccinated Pigs

In order to assess the efficacy of the vaccine combinations, the viralclearance was estimated in BAL fluid, lung parenchyma, and blood ofheterologous virus challenged pigs. In the BAL fluid of groups 4 and 6pigs (100 μg dose) the replicating viral titer was reduced compared togroup 2 animals, but were comparable to other vaccinated groups.However, in the lung homogenate of group 6 pigs the viral titer wassignificantly reduced (FIGS. 29A & E) compared to group 2 pigs. In piggroups 4 and 6 (500 μg dose), the replicating 266 virus was absent inthe BAL fluid (FIG. 29B); but it was completely cleared in the lungs(lung homogenate) of only group 6 pigs (FIGS. 29E & F). Further, ingroups 4 and 6 (100 μg dose) the viral RNA copy numbers were reduced inboth the BAL fluid and lung homogenate (FIGS. 29C & G). While in thesame groups with 500 μg vaccine dose a significant reduction in theviral RNA load was detected compared to group 2 pigs (FIGS. 29D & H).The data indicated that adjuvanted 500 μg of NP-KAg vaccine dose wasefficacious in total clearance of replicating challenged heterologousPRRSV both from the pig lungs and from circulation. In the blood of piggroups 4, 5, and 6 (100 μg dose), a significantly reduced PRRSV titerwas detected compared to groups 2 and 3 at PC 10 (FIG. 291). Again ingroup 6 pigs (500 μg dose), the replicating PRRSV was completely absentin the blood of virus challenged pigs, at all the tested PCs (FIG. 29J).

Microscopic examination of H&E stained lung sections revealed severepneumonic lesions with massive infiltration of mononuclear cells withperivascular cuffing in group 2 and 3 pigs. In contrast, remarkablereduction in inflammatory cells infiltration in adjuvanted 500 μg ofNP-KAg vaccine dose received pigs was observed compared to all othergroups (FIG. 29K).

Adjuvanted NP-KAg Induced High Avidity PRRSV Specific Antibodies

The binding strength of heterogeneous polyclonal antibodies to cognateantigens is defined as avidity (151). At PC 15 in group 6 pigs anincreased avidity of PRRSV specific lung IgA and plasma IgG compared toother test groups were detected (FIG. 31). In particular, significantlyhigher avidity of IgA in BAL and lung homogenate in group 6 pigscompared to other test groups was detected (FIGS. 31A, B & C).Similarly, comparably increased avidity of IgG was observed in lunghomogenate of group 6 pigs compared to other test groups. Surprisingly,avidity of IgG in plasma was high and comparable in pig groups 4 and 6(FIGS. 31E & F). However, at PC 0 and 6, there was no difference in theavidity of IgG in plasma among all the tested groups. Overall, thesedata indicated increased production of high avidity PRRSV specificantibodies in adjuvanted NP-KAg vaccinated pigs both in the lungs andblood.

Adjuvanted NP-KAg Induced Balanced Th1 and Th2 Response Indicating IgGIsotypes

Typically killed vaccines elicit predominantly Th2 responses, butNP-based vaccines drive either Th1-Th2 balanced or Th1 biased responses(138). Therefore, PRRSV specific IgG sub-isotypes were quantified, andin pigs higher IgG1 and IgG2 levels indicate Th2 and Th1 biasedresponse, respectively (149). The levels of PRRSV specific IgG1 and IgG2in plasma (PC 0) and in lung homogenate (PC 15) in pig groups 2 to 5were low and comparable, but their levels were significantly higher inthe plasma (PC 15) of group 6 pigs (500 μg vaccine dose) compared toother groups (FIG. 32A). To assess Th2 or Th1 biased response, the ratioof IgG1/IgG2 was assessed, wherein the ratio of >1 or <1 indicates Th2or Th1 biased response, respectively. In group 6 pigs at PC 15, balancedresponse (ratio close to 1) was detected both in the plasma and lunghomogenate, while groups 3 and 4 pigs had Th2 biased antibody response(FIG. 32B). A similar trend was observed in pigs received 100 μg vaccinedose (FIGS. 32A & B). The data indicated that adjuvanted NP-KAg elicitedTh1-Th2 balanced response.

Discussion

PLGA nanoparticle has the ability to mediate activation, maturation, andantigen presentation by APCs (132). It facilitates sustained release ofvaccine Ags and mediates induction of robust B and T-cell responses(133). PLGA (75:25) was used to entrap UV-inactivated PRRSV (VR2332)Ags, and in vitro protein release profile from entrapped nanoparticleswas consistent with other studies (134). Size and surfacecharacteristics of NPs play an important role in their opsonization andclearance kinetics (135). The NP-KAg was 400-700 nm in diameter, whichwas ideal for uptake by mucosal M-cells and APCs (136). A few changeswere made to NP-KAg preparation compared to an earlier study (123),aimed to enhance the vaccine immunogenicity. NP-KAg were madehydrophilic (by Polaxamer 188) to facilitate easy uptake by APCs (41),sucrose was used as a stabilizer and Mg(OH)₂ to buffer the acidic pHgenerated during hydrolysis of PLGA (137). Such a modified NPs vaccinedelivered intranasally elicits enhanced and prolonged IgG and IgAproduction (138).

PLGA vaccine coadministered with a potent adjuvant elicits protectiveimmune responses (119). Adjuvant M. tb WCL was demonstrated to boost theefficacy of PRRS-MLV (28), and also a single dose of NP-KAg elicitspartial cross-protective immunity in pigs (123). To further potentiatethe efficacy of NP-KAg, the vaccine was coadministered withentrapped/unentrapped M. tb WCL and evaluated various immune correlatesof protection in heterologous PRRSV (MN184) challenged pigs. The resultsindicated that unentrapped M. tb WCL significantly potentiated theimmunogenicity of NP-KAg vaccine (group 6 pigs), as indicated by thefollowing immune correlates: (i) increased PRRSV specific IgG and IgAtiters with enhanced antibody avidity; (ii) balanced Th1 and Th2responses with enhanced VN titers; (iii) increased secretion of Th1(IL-12 and IFN-γ) and Th2 (IL-4) cytokines, with enhanced frequency ofISCs and IFN-γ producing CD4⁺, CD8⁺, CD4⁺CD8⁺ T cells, γδ⁺ 305 T cells,and NK cells, and expanded frequency of APCs; (iv) reduced production ofimmunosuppressive cytokines (IL-10 and TGF-β) and importantly, (v)complete clearance of the replicating challenged virus from both thelungs and blood.

PRRSV primarily infects both interstitial and alveolar Mφs (118), andgreater than 90% BAL cells are Mφs (62), while lung MNCs are rich inboth interstitial Mφs and lymphocytes. CD11c⁺ APCs are present in bothBAL fluid and lung parenchyma, but they differ in their antigenpresentation potential, with the former activates only antigen primed Tcells and the latter activates both naïve and antigen primed T cells(139). Therefore, responses in the BAL fluid, lung homogenate, and lungMNCs were analyzed.

Both at mucosal sites and blood of adjuvanted NP-KAg vaccinated (group6) pigs, significantly enhanced levels anti-PRRSV antibodies weredetected, along with increased antibody avidity and VN titers. Theavidity of polyclonal antibodies in vaccinated or infected animals isshown to be positively correlated with VN titers (140). Consistent witha previous report, avidity of PRRSV specific IgG levels in the plasma ofgroup 6 pigs increased gradually and reached the peak at post-challengeday 15, which was due to gradual domination of high affinity antibodysecreting B cell clones (140). Nanoparticles have been shown to interactwith pathogen recognition receptors on APCs (B cells) leading toaffinity maturation and production of high avidity antibodies (141).Further, the avidity results were correlated with PRRSV VN titers bothat mucosal surfaces (IgA) and lung parenchyma (IgA and IgG), whichcontributes to protection.

Typically killed vaccines elicit predominantly Th2 response, but toefficiently clear virus infected cells balanced Th1 and Th2 responsesare necessary (142). NPs based vaccines drive either balanced or Th1biased responses (138). In group 6 pigs, enhanced and balanced Th1-Th2humoral and cell-mediated immune (CMI) responses were detected. VNantibodies against putative neutralizing epitopes on PRRSV GP5 and Mglycoproteins play an important role in PRRSV clearance (131). In group6 pigs, high levels of cross neutralizing VN titers were detectedagainst challenged PRRSV strain (MN184), and also increased VN titerswere detected against other genetically divergent viruses including thetype I virus strain, indicating the presence of a broadly reactive VNresponse elicited by adjuvanted NP-KAg.

A NK cell is a major innate immune player in antiviral defense. In thegroup 6 pigs, the population of IFN-γ⁺ NK cells was significantlyupregulated. PRRSV suppresses the NK cell activity (95, 143), butadjuvanted NP-KAg appears to rescue the NK cell function. Increasedproduction of IL-12 in the lungs of group 6 pigs can influence the NKcell function. The γδ T cells are present in high frequency in pigs andare involved in both innate and adaptive immunity. Enhanced frequency ofactivated γδ T cells was observed in group 6 pigs. A robust CMI responseis important for complete protection against PRRSV infection (28). Acrucial Th1 cytokine, IFN-γ, is produced by NK cells, γδ T cells, CD4⁺and CD8⁺ T cells, and CD4⁺CD8⁺ T cells. In group 6 pigs, increasedfrequency of IFN-γ secreting lymphocyte and their subsets wereidentified. In addition, significantly reduced production of IL-6 ingroup 6 pigs indicated the absence of inflammatory reaction at PC 15 ingroup 6 pigs. The observed enhanced CMI response in NP-KAg received pigscan be due to PLGA mediated cross-presentation of entrapped Ags to CD8⁺T cells by APCs in vivo (144). An enhanced memory response is associatedwith production of high avidity antibodies and enhanced B and T cellresponses (145). Thus, the immune response results in post-challengedpigs indicate the possible induction of a strong memory response inadjuvanted NP-KAg received pigs.

The importance of immediate availability of unentrapped potent adjuvantto intranasally delivered NP-KAg vaccine was critical to elicit a robustimmune response, because inadequate anti-PRRSV response with partialviral clearance was observed earlier with only NP-KAg vaccine (123), andnow in NP-KAg and NP-M. tb WCL received pigs (group 5). Consistent withthese results, PLGA entrapped Hepatitis B subunit vaccine coadministeredwith an encapsulated adjuvant failed to induce adequate antibodyresponse (146). Further, both vaccine and adjuvant unentrappedformulation (group 4 pigs) elicited robust Th2 biased responses withincomplete clearance of challenged PRRSV, confirming the necessity of aPLGA system to deliver PRRSV KAg. Consistent with the presence of lowPRRSV RNA copy number and absence of replicating virus in adjuvantedNP-KAg vaccinated (group 6) pig lungs, previous reports have shown thatqRT-PCR fails to differentiate infectious and non-infectious virus andthe inactivated PRRSV is relatively stable in the environment (125).Samples with low levels of PRRSV RNA copies failed to replicate in cellculture (56).

The results have shown a potent adjuvant like M. tb WCL can induce abetter cross-protective immunity by PLGA-NanoPRRS. But, M. tb growsslowly in culture and it is a biosafety level-3 (BSL-3) pathogen. Thus,large-scale production of M. tb WCL represents risk, time, and cost.Therefore, a cost-effective potent alternate adjuvant to use withPLGA-NanoPRRS is ideal. Four nonpathogenic species of Mycobacteriumwhose cell wall and protein composition is comparable to M. tb have beenidentified. They include M. smegmatis, M. vaccae, M. alvei, and M.fallax. These four mycobacterial species are fast-growing,non-pathogenic, like M. tb they possess rough colony morphology withcord formation, cell wall characteristics close to M. tb, non-commensalof pigs, BSL1 facility is sufficient to grow them, and available inAmerican Type Culture Collection (ATCC) (112). Moreover, M. smegmatispreparations are used as candidate adjuvants to generate protectiveimmune responses against pathogens (113, 114). In a pilot study,PRRS-MLV and M. smegmatis WCL were co-administered intranasally to pigsand challenged with a heterologous virus; clinically, pigs did not havePRRS symptoms, and immunologically detected enhanced IFN-γ and IL-12production compared to control PRRS-MLV group. M. vaccae preparation wasused as an adjuvant in oral vaccines (115, 116).

A PLGA based delivery system reduces the required vaccine dose byseveral folds, demonstrated recently with a PLGA NPs-based tumor peptidevaccine, wherein a 63 times reduced vaccine dose elicited a comparableresponse (147). Since PLGA NPs are getting global recognition, they arean effective delivery system for inactivated mucosal vaccines, moreovertheir size, contents, and cell targeting properties could be engineered(119). In conclusion, intranasal delivery of a potent adjuvanted PLGAnanoparticle-entrapped inactivated PRRSV vaccine in pigs has thepotential to induce superior cross-protective immunity. Furthervalidation of this vaccine formulation involving large numbers of pigswill envisage its field of application. This strategy can also beapplied to control important human respiratory pathogens.

Example 4 PRRSV VLPs

PRRSV excretes from all the body secretions at low levels or perhapsintermittently in saliva, nasal secretions, urine, milk, colostrum,feces of infected pigs, and also in semen of infected boars [174-179].The PRRSV elicits poor innate and adaptive immune responses in infectedpigs [57, 180, 181, 12], associated with increased immunosuppressiveresponse [77, 182, 183, 48, 95]. As a result in PRRSV infected pigsthere will be incomplete viral clearance and increased susceptibility tosecondary microbial infections [125, 165, 12, 184, 15]. Since the 1990swithin PRRSV genotypes (European-Type I and North American-Type II)there have been constant emergence of several genetically variantsubtypes and strains in the US, resulting in a display of significantdifferences in their pathogenicity in pigs [38]. Therefore, immunityinduced by one PRRSV strain in pigs may provide partial to no protectionto reinfection [185, 186, 117, 187]. Within North American PRRSVisolates genetic variation is from 84-100%, and one such field isolate,MN184, is a virulent and genetically highly variant strain [38].Although many reports have demonstrated satisfactory immunity induced byMLV-PRRS in growing pigs, others reported reversion in virulence ofvaccine virus and transmission to unvaccinated pigs and sows [96, 32,97]; also recombination between vaccine and field strains [188, 88].Thus, prevention of virus transmission from the infected pig herd iscritical to control PRRS. All these limitations made the research ondevelopment of a better cross-protective killed PRRSV vaccine a highpriority.

Activated innate immune response at mucosal sites play a major role inmucosal immunity against enteric and lung infections [189]. Immuneresponses elicited by potent mucosal vaccines is associated withenhanced cytotoxic T-lymphocyte (CTLs) and central memory immuneresponses, targeted to a greater number of viral structural proteins andto many conserved epitopes, resulting in enhanced cross-protectiveimmunity against genetically divergent field viral strains [190, 191,54, 192-194]. Nanotechnology has become one of the important researchendeavors of future vaccinology approach. Nanoparticles offer theadvantage of increasing the potency of drug and vaccine delivery,possess adjuvant properties, and thus improves the vaccine efficacy[60]. Due to inherent ability of APCs to readily phagocytose particulatestructures, PLGA nanoparticle-based vaccines and VLPs prime long-lastingantibody and T cell responses [195-197]. Moreover, due to the nature ofPLGA nanoparticles to protect entrapped vaccine antigens fromproteases-mediated degradation at mucosal surfaces, nanoparticle-basedvaccine delivery is gaining increased attention to induce protectivemucosal immunity. A killed influenza virus vaccine entrapped innanoparticles administered intranasally to mice, rabbits, and pigsinduced protective immunity, and immune responses elicited in pigs byintranasal delivery was significantly higher than intramuscularimmunization [25]. PLGA nanoparticles entrapped vaccines containinghepatitis B, rotavirus, influenza, or parainfluenza viruses delivered tomucosal sites of mice generated protective immunity [54, 25, 55, 102].Biodegradable and biocompatible PLGA nanoparticles are free from anytoxicity and have proved safe to use in humans. PLGA polymers are usedto prepare suture materials and are approved materials by the U.S. Foodand Drug Administration [198, 90, 92]. It has been demonstrated thatcross-protective immunity against PRRSV can be elicited with the help ofPLGA nanoparticle-based PRRSV vaccine. However, for large-scaleproduction of such a particulate vaccine, a cost-effective strategy inbulk preparation of vaccine antigens is essential.

Recombinant baculovirus-mediated insect cell production technologyallows rapid production of virus-like-particles (VLPs) [199]. Suchtechnology has been proved effective for both enveloped andnon-enveloped viruses like calici, influenza, parvo, polyoma, reo,paramyxo, orthomyxo, hepatitis C, Ebola, Marbug, Chikungunya, SARScorona etc., (reviewed in [200, 201]). Co-expression of multiple (2-4)viral surface proteins still results in VLPs that are indistinguishablefrom authentic viral particles [202, 203]. In addition, production of avaccine with DIVA (differentiation of infected from vaccinated animals)potential is possible with VLP based subunit vaccines. Using alreadypublished standard procedures, PRRSV-VLPs can be prepared. VLP basedvaccines have several advantages over the conventional vaccine antigens;they are as follows. (i) Extremely large-quantities of correctly foldedrecombinant proteins can be produced in high density cell-cultureconditions in eukaryotic cells, thus baculovirus system is amenable toscale-up for large-scale vaccine production [204]. (ii) Insect cellsbased vaccine production can be done without the need of mammaliancell-derived supplements, thus the risks of co-culturing opportunisticpathogens is minimized. (iii) There is no threat from baculovirus invaccinated individuals because this virus has a very narrow host-rangein a few species. (iv) VLPs are quite stable with no alteration ofparticle morphology or reduction in immunogenicity even after 9 weeksstorage at room temperature [205]. (v) To further boost the vaccinepotency, PRRSV-VLPs can be entrapped in PLGA nanoparticles and deliveredto pigs, intranasally. (vi) Unlike MARC-145 cell derived semi-purifiedcrude inactivated PRRSV, the dose of VLPs in a Nano-PRRSV-VLP vaccine isquantifiable.

VLPs promote immunogenicity and have been developed for several viralvaccines. Papilloma virus VLP vaccine is licensed for use in humans, anda few other VLP vaccines are in phase I clinical trial [201].Vaccination of pigs using less than 10 μg dose of porcine parvo virusVLPs in a water-in-mineral oil emulsion adjuvant was found to be highlyimmunogenic, and also efficient in preventing trans-placental virustransmission and number of reproductive failures in gilts [206]. TheVLPs of hepatitis B surface antigen entrapped in PLGA-nanoparticles havebeen found to be effective in trans-mucosal delivery of the vaccine,representing an approach for the delivery of VLPs [207]. Also PLGAnanoparticle-based vaccine delivers the associated VLPs to the immunesystem in a controlled manner for up to 14 days, without any compromisein its immunogenicity [207]. Delivery of PLGA nanoparticle-entrappedPRRSV-VLPs vaccine can elicit an adequate cross-protective immuneresponse in pigs, indicated by enhanced-neutralizing antibody titers andincreased clearance of challenged virulent heterologous PRRSV.

PRRSV Isolates for Candidate Vaccine Construction

Type I virus, SD01-08, and Type II virus, SD09-28, cab be used. TheSD01-08 isolate represents a group of emerging Type I PRRSV in NorthAmerica, which has been used in our previous studies for vaccinedevelopment [170, 171]. The PRRSV SD09-28 was originally obtained in2009 from a PRRSV-infected farm, which represents current fieldcirculation strains (contains 1-8-4 RFLP pattern in ORFS).

Cloning of PRRSV Surface Protein Genes and Generation of PRRSV-VLPs inBaculovirus System

Recombinant baculovirus protein expression system is an establishedsystem [208, 209]. Four important PRRSV surface protein genes, GP3, GP4,GP5, and matrix proteins can be amplified by RT-PCR from the viral RNAand subcloned into a baculovirus transfer vector ‘pAcAB4’ (BDBiosciences, BD BAculoGold™ Cat#554770) using Rapid Ligation Kit(Promega Corp.). Earlier, co-expression of 3 to 4 viral capsid proteinsto make VLPs of influenza virus [210] and bluetongue virus [211] ininsect cells have been demonstrated. To generate PRRSV-VLPs, pAcAB4vector containing PRRSV genes constructs in the correct orientation canbe co-transfected to Sf9 insect cells using Linearized Baculovirus DNATransfection Kit (BD Biosciences, BD BaculoGold™). To detect generatedPRRSV-VLPs, media from the transfected Sf9 cells can be examined usingPCR for the presence of PRRSV nucleotide sequences in the recombinantbaculovirus. Further, PRRSV-VLPs can be visualized with the help ofTransmission Electron Microscope (TEM) (Hitachi S-3500N), by comparingside-by-side with sucrose purified wildtype PRRSV.

Preparation of Nano-PRRSV-VLPs

PLGA nanoparticle-based PRRSV vaccine can be prepared as previouslydescribed [60, 198]. Briefly, PRRSV-VLPs of both the PRRSV strains canbe entrapped in PLGA nanoparticles by double emulsion method,separately. The following chemicals can be used to make thenanoparticles hydrophilic and stable, such as, poloxamer 188, sucrose,Mg(OH)₂, polyvinyl alcohol [212, 41, 213]. Briefly, 15% of PLGA (75/25)can be dissolved in dichloromethane and PRRSV-VLPs, and the mixture canbe homogenized by sonication, then added to aqueous solution ofpolyvinyl alcohol, and again homogenized. Nano-PRRSV-VLPs can be stirredat room temperature, washed, freeze-dried, and stored at 4° C. To use oncontrol pigs, empty PLGA nanoparticles can be prepared by a similarmethod.

Characterization of Nano-PRRSV-VLPs:

The concentration of VLPs in Nano-PRRSV-VLPs can be determined asdescribed previously [41] using a BCA protein assay kit (Biorad, CA).Morphology (size and shape) of Nano-PRRSV-VLPs can be determined bycoating the freeze-dried vaccine powder with gold-platinum under vacuumwith the help of an ion coater and examined using TEM at 10 KV. Further,phagocytosis of Nano-PRRSV-VLPs by pig alveolar MΦs can be determined bytreating BAL cells with freeze-dried Nano-PRRSV-VLPs or PRRSV-VLPs andimmunostained for confocal microscopy (Leica confocal microscope).Further, to determine Nano-PRRSV-VLPs induced activation of alveolarMΦs, treated BAL cells can be subjected to phenotypic analyses usingFACS Aria II (BD Biosciences) flow cytometer.

Evaluation of Efficacy of Nano-PRRS-VLPs Vaccine in Pigs

Conventional 4-6 weeks old healthy pigs can be procured (n=35) from aSPF swine herd free from PRRS at The Ohio State University, OARDC. Serumsamples collected before starting the study can be confirmed negativefor PRRSV antibody. Pigs can be unvaccinated (group 1), vaccinated withempty nanoparticles (group 2), equal amounts of Nano-PRRS-VLPs of boththe viral genotypes (group 3&4), PRRS-VLPs (groups 5&6), or with acommercial killed PRRSV vaccine (group 7), inoculated twice at 2 weeksinterval (Table 4). On day post-vaccination (DPV) 28, pigs can bechallenged using a heterologous PRRSV strain, MN184 [38, 214] (1×10⁶pfu/pig). Animals can be monitored daily for clinical disease and rectaltemperature, and on every 3rd day record body weight. Serum samples canbe collected at different DPV as indicated (Table 4), aliquoted andstored at −70° C. Pigs can be euthanized on DPV 42 (2 weekspost-challenge) and the lungs and lymph nodes can be examined for grosslesions. The lung tissue samples can be collected in neutral bufferedformalin for histological studies. During necropsy blood,bronchoalveolar lavage (BAL) fluid, tracheobronchial lymph nodes (TBLN),tonsils, and lung tissue can be collected for virus detection; and alsoto isolate PBMC, TBLN mononuclear cells (MNC), BAL cells, and lung MNCfor immunological studies [47, 162, 95].

TABLE 4 Evaluation of the efficacy of Nano-PRRS-VLPs in pigs Groups 2-6:two doses of vaccine, 1^(st) dose intranasally, and 2 weeks later the2^(nd) dose (booster) by intramuscular route. Group 7: administered byintramuscular route. Vaccine PRRSV combination Vaccine dose challengeCollection of Pigs (n = 5 per pig MN184 blood samples group pigs/group)Two doses (DPV 21) (DPV) 1 Mock None No 0, 14, 28, 35, 42 Challenge 2Mock Empty nanoparticles Challenge 0, 14, 28, 35, 42 3 Nano-PRRS- 50μg/dose/pig Challenge 0, 14, 28, 35, 42 VLPs 4 Nano-PRRS- 250μg/dose/pig Challenge 0, 14, 28, 35, 42 VLPs 5 PRRS-VLPs 50 μg/dose/pigChallenge 0, 14, 28, 35, 42 6 PRRS-VLPs 250 μg/dose/pig Challenge 0, 14,28, 35, 42 7 Commercial As per recommen- Challenge 0, 14, 28, 35, 42killed dation of the PRRSV manufacturer vaccineDetection of Immune-Correlates of Protection and Cross-ProtectiveImmunityDetermine Humoral Immunity

To compare humoral immune responses, all the serum samples (Table 4) canbe evaluated using the IDEXX HerdChek® PRRS 3XR ELISA and virusneutralization assay [179, 165, 164]. To assess cross-protectiveneutralizing antibody titer induced in Nano-PRRS-VLPs vaccinated pigs, apanel of six field isolates can be used in the assay. Twenty three fieldPRRSV isolates have been collected at the SD Animal Disease DiagnosticLaboratory since 2010. The GP5 sequence data can be entered into thecurrent PRRSV database (prrsvdb.org/) for phylogenetic analysis usingthe method described previously (127). Six isolates that represent thecurrent circulating field strains (most recently evolved PRRSV) can beselected, and used in the assay to evaluate the depth of protectioninduced of the vaccine.

Quantification of Viral Load in Tissues and Viremia

To quantify PRRSV RNA and to determine the viral load, serum samples andtissue samples (lungs, tonsils, and TBLN) can be analyzed byquantitative RT-PCR and cell culture immunofluorescence assay [214, 163,215].

Phenotypic Analysis of Immune Cells

TBLN-MNC, BAL cells, lung MNC, and PBMC can be immunostained todetermine the frequency of both lymphoid and myeloid immune cells byflow cytometry [48].

Cytokines Analysis:

Serum and BAL samples can be analyzed for cytokines: innate (IFN-α);pro-inflammatory (IL6); T-helper 1 (Th1) (IFN-γ and IL-12); andimmunosuppressive (IL-10 and TGF-β by ELISA [48].

NK Cell-Cytotoxicity Assay

Innate NK cell-mediated cytotoxicity can be determined using lung MNCand PBMC as a source of NK cells (effectors) against K-562 target cellsas described previously [12].

Results

Results of the study can be analyzed using a non-parametric statisticaltest (GraphPad prism 5). Based on clinical parameters, viral load,frequency of different immune cells, cytokines profile, and NK cellfunction, the immune correlates of protection in vaccinated, viruschallenged animals can be determined.

Varying degrees of cross-neutralizing antibody response against a panelof field PRRSV isolates in pigs vaccinated with Nano-PRRSV-VLPs can bedetected. Vaccinating with two doses of Nano-PRRSV-VLPs vaccine canresult in better virus clearance from the serum and lungs of pigscompared to results showed in preliminary data. Also in vaccinated viruschallenged pigs, an increased PRRSV specific adaptive immune responseassociated with increased production of cytokines and upregulatedexpression of CD4 and CD8 markers on lymphocytes present at bothsystemic and mucosal sites can be detected. Enhancing the efficacy ofPRRS-VLPs alone or as a candidate vaccine co-administered with potentadjuvants, similar to a published vaccine studies using parvovirus VLPsin pigs, can be achieved [206].

Example 5 Role of UEA in PLGA-NanoPRRS Vaccine

NP-KAg can be targeted to M cells present in the pig upper respiratorytract. Earlier reports have showed that UEA entrapped (inside) PLGAnanoparticle vaccine, inoculated intranasally to mice significantlyenhances the production of specific SIgA. Hypothetically, surfaceanchored UEA in nanoparticle vaccine should be better than entrapped UEAin delivery of its cargo to M cells. In humans, nanoparticle-mediatedtargeted delivery of drugs and biomarkers are achieved by surfaceanchoring the nanoparticles with a targeting molecule (152, 153), whichalso significantly reduces the required dose. Therefore, to enhance theefficacy of NP-KAg vaccine and to make it cost-effective both strategieswill be tested in the pig system.

Based on previous results (123) and results by others (154-158);undoubtedly, the NP-KAg vaccine has the potential to providecross-protective immunity against PRRS. Results of in vitro studies canhelp understand the role of UEA in targeted delivery of NP-KAg to Mcells. This study can help reduce the vaccine dose and further enhancethe cross-protective efficacy to PRRSV. In addition, studies with NP-KAgindicate a potent adjuvant can be used to elicit better immune responsesand viral clearance both from the lungs and circulation. The adjuvant M.tb WCL can be replaced with another candidate potent adjuvant. Thesestudies analyze the adjuvant effects of four selected nonpathogenicmycobacterial species derived WCL, coadministered intranasally withNP-KAg-UEA.

Experimental Methods

Animal Groups and Inoculations

PRRSV antibody free 4-6 weeks old SPF pigs (n=60, 6 pigs per group) willbe randomly assigned into one of the 10 groups. As per the statisticalpower analysis (ebook.stat.ucla.edu/cgi-bin/engine.cgi) six pigs/groupcan provide a Power of at least 0.8 (α=0.05). Pigs can be unvaccinated(mock—group 1) or vaccinated with sham nanoparticles (group 2), killedPRRSV (group 3&4), or with indicated NP-KAg preparations (group 5-10)(Table 5). Pigs can be vaccinated with 5×10⁵ TCID₅₀ (˜100 μg) (groups 3,5, 7, & 9) or 2.5×10⁶ TCID₅₀ (˜500 μg) (groups 4, 6, 8 & 10) per pigdose of indicated NP-KAg vaccine formulations, coadministered with M. tbWCL (1 mg/pig), twice, intranasally at two weeks interval. Note that,100 and 500 μg of vaccine dose refers to protein equivalent of NP-KAg.Just before vaccination freeze-dried vaccine and adjuvant can bereconstituted in PBS, and the required amount for each vaccine dose canbe mixed and the quantity adjusted to 4 ml in PBS. Pig groups 2 to 10can be challenged using the virulent heterologous PRRSV stain MN184 (38)(5×10⁵ TCID₅₀ in 4 ml) at two weeks post-booster [28 dpv (daypost-vaccination], intranasally; while the mock group can receive equalamount of the MARC-145 cell culture supernatant.

TABLE 5 Pig (6 pigs/Gp) Groups Pig groups (n = 60) 1 Mock 2 Shamnanoparticles 3&4 Killed PRRSV 5&6 NP-KAg 7&8 NP-KAg -surface UEA  9&10NP-KAg - entrapped UEAClinical Monitoring, Blood, and Tissue Sampling

Pigs can be monitored daily after PRRSV challenge for disease symptomssuch as respiratory distress, cough, and food intake. Body temperaturecan be recorded every day during first 7 days, and the body weightmeasured every week until dpv 56. Blood samples can be collected on 0,3, 7, 14, 21, 28, 28, 35, 42, 49, and 56 dpv in EDTA, and the plasma canbe aliquoted and stored at −70° C. Pigs can be euthanized at four weekspost-challenge (56 dpv), and the lungs and lung drainingtracheobronchial lymph nodes (TBLN) can be scored for macroscopiclesions (159). Lung samples can be collected in neutral bufferedformalin, sectioned (5 μm) and examined after subjecting to H&E andimmunohistochemistry staining for microscopic lesions (121). The slidescan be scored by a board certified veterinary pathologist, withoutproviding the sample history. Samples of the lungs, tonsils, and TBLNcan be frozen to determine the PRRSV titer later. Blood samplescollected in anticoagulant solution and lung tissue samples collected inDMEM can be processed on the same day to isolate respective mononuclearcells (160, 161, 162). Lung lysates prepared from the lung samples andBAL fluid can be aliquoted and stored at −70° C. (12, 48).

Quantification of Viral Load

PRRSV load and titer in plasma and tissue samples (lungs, tonsils, andTBLN) can be quantified by estimating the viral RNA by RT-PCR (163), andalso by indirect immunofluorescence assay to determine the replicatingviral titer (48) (FIG. 35)

Evaluation of Innate, Humoral, and Cell-Mediated Immune Responses

To analyze cross-protective immune response six genetically variantPRRSV isolates can be selected based on the GP5 sequence data thatrepresent the current circulating PRRSV in the field from the database(prrsvdb.org). Semipurified crude PRRSV Ags prepared from six fieldPRRSV isolates can be used to stimulate PBMCs and lung MNCs to analyzeIFN-γ⁺ lymphocytes frequency by ELISPOT assay (128) (FIG. 36). Ascontrols, cells stimulated with PHA (10 μg/ml) or unstimulated can beincluded. Plasma samples can be evaluated using the IDEXX HerdChek® PRRS2XR ELISA for viral antibodies (48). Cross-protective VN titers inplasma samples collected at 0, 28, 42, and 56 dpv and lung lysatescollected at dpv 56 can be determined against six PRRSV field isolates(164, 165) (FIG. 37). Avidity of PRRSV specific IgA and IgG in BALfluid, lung lysates, and plasma samples will be determined (166, 167)(FIG. 38). Plasma samples collected at dpv 0, 3, 7, 14, 21, 28, 28, 35,42, 49, and 56 and lung lysates will be analyzed for: innate (IFN-α,IFN-β); pro-inflammatory (IL1β, IL6, and TNFα); T-helper 1 (Th1) (IFN-γand IL-12); Th2 (IL-4); and immunosuppressive (IL-10 and TGF-β)cytokines by ELISA (12).

Adjuvant Effects of Nonpathogenic Mycobacterial Adjuvants to NP-KAg-UEAVaccine

Experimental Methods

Production of WCL

Four selected non-pathogenic Mycobacterium species (Table 6) can begrown in liquid medium as recommended by ATCC, and WCL can be preparedas described previously (168). Briefly, live bacteria can be harvestedand washed twice using PBS (pH 7.4) and suspended (2 g/ml) in PBScontaining 8 mM EDTA, proteinase inhibitors, DNase, and RNase. Cells canbe disrupted by using the Bead Beater until approximately 90% breakageis obtained (monitored by acid fast staining), centrifuge at 3,000×g topellet unbroken cells and insoluble cell wall components, and thesupernatant (WCL) can be harvested. The protein content and endotoxinlevels in the WCL can be quantified using the kits, and the aliquotswill be stored at −70° C. The mycobacterial WCL preparation containswater-soluble proteins, lipids, and carbohydrates.

TABLE 6 Pig (6 pigs/Gp) Groups Pig groups (n = 60) Adjuvant 1 Mock 2Sham nanoparticles 3&4 NP-KAg -UEA M. smegmatis 5&6 NP-KAg -UEA M.vaccae 7&8 NP-KAg -UEA M. alvei  9&10 NP-KAg -UEA M. fallaxAnimal Groups and Inoculations

PRRSV antibody free 4-6 weeks old SPF pigs (n=60, 6 pigs/group) can berandomly assigned into one of the 10 groups (Table 6). Pigs can beunvaccinated (mock—group 1), vaccinated with Sham nanoparticles, orvaccinated with 5×10⁵ TCID₅₀ (˜100 μg) per pig dose (groups 3, 5, 7, &9), or 2.5×10⁶ TCID₅₀ (˜500 μg) per pig dose (groups 4, 6, 8 & 10) ofNP-KAg-UEA vaccine, coadministered intranasally with indicatednonpathogenic Mycobacterium species derived WCL (groups 3 to 10), 1mg/pig, twice, at two weeks interval. Just before vaccination,freeze-dried vaccine and indicated adjuvant preparations can bereconstituted in PBS, and the required amount for each vaccine dose canbe mixed and the quantity adjusted to 4 ml in PBS. Pig groups 2 to 10can be challenged with the virulent heterologous PRRSV strain MN184 (38)(5×10⁵ TCID₅₀ in 4 ml) intranasally, at two weeks post-booster [28 dpv(day post-vaccination]; while the mock group can receive the cellculture supernatant. Monitoring of clinical signs, collection of bloodand tissue samples, quantification of viral load, and evaluation ofhumoral, innate, and cell-mediated immune responses can be performed asdescribed above.

The Breadth of Cross-Protective Immunity in AdjuvantedNanoparticle-PRRSV Vaccinated Pigs Against Different AntigenicallyDivergent Viruses

This study can determine how the adjuvanted NP-KAg-UEA vaccine helps ininduction of better cross-protective immunity, measured by clinicalsymptoms, viral clearance, and by immune correlates in growing pigs. Inpregnant sows, whether the vaccine formulation could reduce the verticaltransmission of the challenged virus to piglets can be investigated.

Experimental Methods:

Animal Groups and Inoculations

PRRSV antibody free 4-6 weeks old SPF pigs (n=66) and pregnant sows at55 days of gestation (n=9) can be randomly assigned into one of theindicated groups (Table 7). Pigs can be unvaccinated (mock—group 1),vaccinated using sham nanoparticles, or with NP-KAg-UEA vaccine (Table7a&b), coadministered intranasally with a selected non-pathogenicMycobacterium species WCL (1 mg/pig), twice at two weeks interval. Justbefore vaccination, freeze-dried vaccine and adjuvant preparations canbe reconstituted in PBS and the required amount can be mixed andadjusted to 4 ml in PBS.

TABLE 7a (6 pigs/Gp) Challenge GP Pig groups (n = 66) PRRSV 1 Mock 2Sham Nanoparticles VR2332 3 Sham Nanoparticles 10-398 4 ShamNanoparticles SD03-08 5 NP-KAg -UEA VR2332 6 NP-KAg -UEA 10-398 7 NP-KAg-UEA SD03-08 9 PRRS-MLV VR2332 10 PRRS-MLV 10-398 11 PRRS-MLV SD03-08

TABLE 7b (3 sows/Gp) GP Sow groups (n = 9) Challenge 1 Mock 2 ShamNanoparticles 10-398 3 NP-KAg -UEA 10-398

As a control, pig groups 9 to 11 can be vaccinated using PRRS-MLV as permanufacturer recommendations. Pigs groups 2 to 11 (Table 7a) and groups2 and 3 (Table 7b) can be challenged with an indicated PRRSV strain, twoweeks after booster (28 dpv) (5×10⁵ TCID₅₀ in 4 ml), intranasally; whilethe mock group can receive equal amount of MARC-145 cell culturesupernatant.

The challenge viral strain, PRRSV 10-398 (D6) (accession #10-16734)contains 1-4-4 RFLP pattern in ORFS (169), and it was isolated in 2010from an infected sow, represents an extremely virulent current fieldstrain in the state of Ohio and other states, which killed 10% ofinfected sows. The PRRSV strain SD01-08 represents an emerging Type IPRRSV in North America (170, 171). The homologous virus (VR2332 strain)(164, 172) can also be included. Monitoring of clinical signs,collection of blood and tissue samples, quantification of viral load,and evaluation of humoral, innate, and cell-mediated immune responses ingrowing pigs can be performed as described above.

Clinical Monitoring, Blood, and Tissue Sampling of Pregnant Sows

Pregnant sows can be monitored daily for clinical signs and bloodsamples will be collected at gestation day 60 (dpv 0), 88, and on theday of euthanasia. Sows and neonates can be euthanized and the samplescollected as described earlier (173). Briefly, between 109 and 112 daysof gestation, sows can be euthanized and the uterine horns can beimmediately necropsied and number of live and stillborn fetuses can berecorded. Blood samples can be collected from each fetus and plasma canbe aliquoted and stored at −70° C. Both maternal and fetal tissues(TBLN, tonsil, and lung) can be collected and stored in formalin forhistological studies, and also similar tissue samples collected in RNAlater (Ambion) can be analyzed for PRRSV RNA and cytokine mRNA byqRT-PCR.

Statistical Analyses

Data analyses can be carried out using SAS. Comparisons of viral load,cytokine levels, and VN titers can be performed by analysis of variance(ANOVA), with Duncan's multiple comparison tests used when the ANOVAtest indicates significant difference. Comparisons of antibody responsecan be made using the Kruskal-Wallis non-parametric test.

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What is claimed is:
 1. A composition comprising an inactivated porcinereproductive and respiratory syndrome virus (PRRSV) entrapped within ananoparticle, wherein the composition further comprises an adjuvant,wherein the adjuvant comprises a Mycobacterium lysate.
 2. Thecomposition of claim 1, wherein the adjuvant is a Mycobacteriumtuberculosis whole cell lysate.
 3. The composition of claim 1, whereinthe adjuvant is selected from the group consisting of a Mycobacteriumsmegmatis whole cell lysate, Mycobacterium tuberculosis whole celllysate, and Mycobacterium vaccae whole cell lysate.
 4. The compositionof claim 1, wherein the nanoparticle is immunogenic.
 5. The compositionof claim 1, wherein the inactivated PRRSV is entrapped within thenanoparticle by a water/oil/water emulsion method.
 6. The composition ofclaim 1, wherein the nanoparticle is poly(lactide co-glycolide) (PLGA).7. The composition of claim 6, wherein the PLGA is 50/50 PLGA.
 8. Thecomposition of claim 1, in a ratio of about 180 mg of PLGA to about 5 mgof inactivated PRRSV.
 9. The composition of claim 1, wherein theinactivated PRRSV is the MN184 strain of PRRSV.
 10. The composition ofclaim 1, wherein the inactivated PRRSV is inactivated by UV light orbinary ethylenimine.
 11. A vaccine comprising a composition of claim 1in a carrier.
 12. A method of eliciting an immune response against PRRSVin a pig comprising administering to said pig the vaccine of claim 11.13. The method of claim 12, wherein the immune response is protectiveagainst PRRSV infection.
 14. A method of reducing reproductive orrespiratory failure in a pig comprising administering to the pig thevaccine of claim 11 to.
 15. A method of stimulating an immune responsein a pig comprising: administering to said pig the vaccine of claim 11.16. The method of claim 12, wherein the vaccine is administered at adose of between 100 ug/pig and 500 ug/pig.
 17. The method of claim 12,wherein the vaccine is administered in a single dose.
 18. The method ofclaim 12, wherein the vaccine is administered in two doses.
 19. Themethod of claim 18, wherein the two doses are administered at about twoweek intervals.
 20. The method of claim 12, wherein the vaccine isadministered intranasally.